bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric oxide synthase inibition and thermoregulatory control of local sweat rate in exercising humans
Authors: Mack Gary W. , Welch Garrett , Foote Kristopher M.
Published: June 2021

The role of nitric oxide (NO) in modulating human thermoregulatory sweating was examined in 10 subjects (5 male/5 female). Three intradermal microdialysis probes were placed in the dorsal forearm. The control site was perfused with 0.9% saline while the two remaining sites were perfused with a nitric oxide inhibitor: 10 mM NG‐nitro‐L‐arginine (L‐NAME) or 10 mM NG‐monomethyl‐L‐arginine (L‐NMMA). Local sweat rate (SR) and skin blood flow (SkBF) were monitored directly over the path of the intradermal microdialysis probe while arterial blood pressure was measured in the opposite arm non‐invasively. Thermoregulatory sweating was induced by cycle ergometer exercise (60% VO2max) in a warm environment (30°C). Thermoregulatory control of SR was described by the following four parameters: resting sweat rate (SRREST), the increase in esophageal temperature required to initiate a significant rise in SR (ΔTesTHRESHOLD), the slope of the initial linear rate of rise in SR per unit rise in Tes (SR‐ΔTesSLOPE), and peak SR (SRPEAK). In general, NOS inhibition attenuated thermoregulatory sweating during dynamic exercise in a warm environment as shown in the Table below.

L‐NMMA appeared to be more effective, than L‐NAME, in reducing the SR response. We conclude that NO has a subtle but physiologically active role in thermoregulatory control of sweating. (supported by NIH‐RO1‐HL039818)
Modulatory effects of GABA in the Paraventricular Nucleus of conscious rats subjected to exercise training
Authors: Martins‐Pinge Marli Cardoso , de Abreu Samantha Bagolan , Lenhard Adriane , Mehanna Aida , Hasser Eileen M
Published: June 2021

The paraventricular nucleus (PVN) of the hypothalamus is an important site for autonomic and neuroendocrine regulation, where gamma‐aminobutyric acid (GABA) system play an important role. The central mechanisms underlying modulatory effects of exercise training, have yet to be characterized. The aim of this study was analyze the cardiovascular effects of microinjection of bicuculine (BIC), muscimol (Musc), into the PVN of conscious rats submitted to swimming training (ST). After the ST protocol, adult male Wistar rats (250 to 300 g) were anesthetized for implantation of guide cannulas to PVN. After 5 days, a femoral artery and a femoral vein were catheterized for recording mean arterial pressure (MAP) and drugs injection. 24 hours later, unilateral microinjection of BIC (n=8) in the control rats (C) produced an increase in MAP (23±3 mmHg) and HR (93±12 bpm). After ST (n=9) the cardiovascular effects of BIC were attenuated for MAP and did not change for HR (DMAP= 15±2 mmHg; DHR= 88±19 bpm). A bilateral microinjection of Musc in C rats induced a decrease in MAP and HR (DMAP= −14±1 mmHg; DHR=−95±18 bpm). After ST Musc promoted a higher decrease in MAP and no alterations in HR (DMAP= −28±4 mmHg, DHR=−54±12 bpm). Our data showed that ET seems to enhance GABA tonic inhibition in the PVN and/or a blunted effect in the excitatory system to regulate cardiovascular function, which are in line with a decreased sympathetic activity.
Energy efficiency and substrate partitioning in individuals with atherogenic lipoprotein profile
Authors: Faghihnia Nastaran , Siri Patty W. , Krauss Ronald M. , Brooks George A.
Published: June 2021

Individuals with an atherogenic lipoprotein profile (ALP), characterized by increased triglyceride, low HDL, and small dense LDL particles, were found to require greater caloric restriction to achieve the same amount of weight loss compared to unaffected subjects (Krauss, R, unpublished data, 2006).

PURPOSE:

We sought to determine the physiological basis for the greater caloric restriction required for weight loss in subjects with ALP.

METHOD:

We studied seven men with ALP (ALP+) and six unaffected controls (ALP−) during rest (30 min), exercise (20 min) and postexercise recovery (30 min) by indirect calorimetry to determine energy efficiency and substrate partitioning after an overnight fast. Delta efficiency (DE) was calculated as Δ work accomplished/Δ energy expended between workloads of 10, 50, 100, and 150 watts.

RESULTS:

ALP+ had higher DE than ALP− at 50–10 watts (P < 0.05). Respiratory exchange ratios (RER) were significantly (P < 0.05) elevated in ALP+ vs ALP– during rest (0.86 +/− 0.01 vs 0.81 +/− 0.02), exercise (0.99 +/− 0.02 vs 0.92 +/− 0.02) and postexercise recovery (1.1 +/− 0.05 vs 0.95 +/− 0.05, respectively).

CONCLUSION:

Increased efficiency at low exercise intensity and reduced lipid oxidation during rest, exercise and postexercise recovery in ALP+ may explain their increased resistance to weight loss. Supported by NIH grant AR 42906, National Dairy Council, and Atherosclerosis Research at CHORI.
The pulmonary microvascular endothelial cell glycocalyx includes sialic acid important for endothelial barrier function
Authors: Cioffi Eugene A. , Cioffi Donna L. , Stevens Troy
Published: June 2021

The endothelium forms a semi‐permeable barrier between constituents of the blood and underlying tissue. The endothelial glycocalyx consists of glycoproteins, glycolipids and proteoglycans which coat the cell surface. The carbohydrate network that contributes to the glycocalyx is very complex, and to date, the structure of these complex carbohydrates and their role in endothelial barrier function are unknown. We have begun to probe the molecular identities of endothelial cell surface carbohydrates in a systematic way. Initial studies examined the presence of terminal sialic acid residues. Neuraminidase is an enzyme that cleaves sialic acids from carbohydrate chains. Cultured rat pulmonary microvascular endothelial cells (PMVECs) were treated with neuraminidase from Clostridium perfringe (1 unit) for two hours. Neuraminidase treated cells exhibited loss of cell‐cell adhesion as noted by the formation of interendothelial cell gaps, and in a scratch‐wound assay neuraminidase treated cells were slower to reseal as compared to control. These observations suggest that the PMVEC glycocalyx contains terminal sialic acid residues that play a role in endothelial barrier function. Supported by HL60024.
Podoplanin is required for human lymphatic endothelial cell migration in response to VEGF stimulation
Authors: Navarro Angels , Perez Ricardo E , Rezaiekhaligh MO , Mabry Sherry M , Ekekezie Ikechukwu I
Published: June 2021

Podoplanin, a transmembrane protein with expression in endothelial cells restricted to those of lymphatics, has been associated with increased tumor migratory and invasive behavior. However, its role, if any, in endothelial cell migration is unknown.

METHODS: Confluent monolayers of siRNA‐podoplanin silenced versus control human lung lymphatic microvascular endothelial cells (HMVEC‐LLy) were scratch‐wounded and cell migration analyzed in basal media (0.5% FBS), VEGF‐containing media (10 ng/ml in basal media) or complete media (5% FBS and growth factors). Pictures were acquired at regular intervals over 2–3 days. Cell polarization was assessed 2–3 hours after wounding by immunocytochemistry for α‐tubulin and pericentrin.

RESULTS: By 48 hours, wounds in control HMVEC‐LLy exposed to either 10 ng/ml VEGF or complete media are 100% healed versus only 40% at 72hrs for siRNA‐podoplanin HMVEC‐LLy. Moreover, reorientation of microtubule‐organizing center (MTOC) towards the front of the cell in response to VEGF is observed to be impeded in these cells.

CONCLUSION: Podoplanin is required for a full migratory response to VEGF in HMVEC‐LLy. Complete reorientation of the MTOC in response to VEGF, necessary for directional migration, is achieved only if the cells express podoplanin.

Funded by: Children's Mercy Hospital
Thrombin activates calcium independent phospholipase A2 (iPLA2) in lung microvascular endothelial cells
Authors: Rastogi Prerna , McHowat Jane
Published: June 2021

The alveolar‐capillary barrier comprises of type 1 pneumocytes of the alveolar wall, endothelial cells of the capillaries and the basement membrane. Its function is to prevent blood entering the alveoli and to facilitate efficient gaseous exchange. Any disruption in the lining of the lung microveasculature is potentially deleterious. This study examines thrombin stimulation of human lung microvascular endothelial cells (HMVEC‐L) and the resultant increase in iPLA2 activity. Incubation of HMVEC‐L with thrombin resulted in a fourfold increase in iPLA2 activity that remained elevated for up to 60 minutes. Pretreatment of HMVEC‐L with bromoenol lactone (BEL, 2μM, 10 min, a selective iPLA2 inhibitor) but not with PX‐18 (2μM, 10 min, an sPLA2 inhibitor), or AACOCF3 (2μM, 10 min, a cPLA2 inhibitor) attenuated thrombin‐activated iPLA2. Activation of iPLA2 resulted in a rapid increase in arachidonic acid and PGI2 release that was also inhibited by BEL pretreatment. Thrombin stimulation also resulted in a four‐fold increase in neutrophil adherence to the apical surface of HMVEC‐L that was inhibited by BEL pretreatment of HMVEC‐L and pretreatment of the neutrophils with a PAF receptor antagonist. Taken together, these results demonstrate that iPLA2 inhibition has the potential to block the several inflammatory responses produced by thrombin in the lung microvasculature.

Supported by AHA, Pre‐Doctoral Fellowship (PR)
Store‐operated hypoxic but not hypercapnic calcium rise is reversed in rat glomus cells by 2‐APB
Authors: Lahiri Sukhamay , Baby Santhosh Mukadiyil , Roy Arijit
Published: June 2021

We studied calcium responses in the enzymatically separated rat carotid body glomus cells. Store‐operated hypoxic calcium rise in glomus cells was reversed by 2‐APB whereas those by hypercapnia and by high extracellular [K+] were not. 2‐APB and mitochondrial inhibitions completely overlapped, pointing to the same mechanism of effects. Also, ATP elevated the intracellular calcium in normoxia due to influx and to release from the intracellular stores, both of which were inhibited by 2‐APB. This distinction as revealed by 2‐APB is unique and is due to possible involvement of phosphorylation mechanisms in hypoxia. Also, the store‐operated calcium channels in the endoplsmic reticulum where IP3 receptors reside and is reacted with 2‐APB is critical for the hypoxic inhibitory response. Hypercapnia and high extracellular K+ have no access to this region of endoplasmic reticulum, and hence have no effects. The specific inhibition of calcium rise by 2‐APB is yet a novel example of cellular calcium signaling. (Supported by NIH grant R37‐HL‐43413).
Nox1‐derived reactive oxygen species (ROS) mediate thrombin‐induced influx of calcium in vascular smooth muscle cells (SMC)
Authors: Zimmerman Matthew , Takapoo Maysam , Damahalli Jagadeesha , Stanic Bojana , Banfi Botond , Miller Francis , Bhalla Ramesh
Published: June 2021

Mechanisms by which NADPH oxidase‐derived ROS modulate cell signaling pathways are poorly understood. Using SMC cultured from aorta of Nox1 null and littermate control (WT) mice, we tested the hypothesis that ROS generated by Nox1, a catalytic subunit of NADPH oxidase, modulate thrombin‐induced increases in intracellular calcium ([Ca2+]I). In WT SMC, thrombin (1 U/ml) rapidly increased [Ca2+]I with a peak of 1100±70 nM Ca2+ (n=89 cells), as measured by fura‐2 fluorescence ratio imaging, which was inhibited by antisense to Nox1. Similarly, thrombin‐induced increase in [Ca2+]I was markedly attenuated in Nox1 null SMC (620±70 nM Ca2+, n=114 cells, p<0.05). Investigation into potential mechanisms by which Nox1‐derived ROS modulate [Ca2+]I showed that thrombin‐induced generation of inositol trisphosphate and the release of intracellular Ca2+by thapsigargin were similar in WT and Nox1 null SMC. In contrast, bathing cells in Ca2+‐free media significantly reduced the thrombin‐mediated increase of [Ca2+]I in WT SMC, whereas the response in Nox1 null SMC was unchanged. Sodium vanadate, an inhibitor of protein tyrosine phosphatases (PTPs), normalized the thrombin‐induced increase of [Ca2+]I in Nox1 null SMC. These data suggest that inhibition of PTPs by Nox1‐derived ROS mediate influx of extracellular Ca2+ in SMC, and identify a novel signaling mechanism that may have a role in vascular disease.
Intermittent hypoxia in rats augments myogenic contraction in mesenteric arteries.
Authors: Jackson‐Weaver Olan , Bosc Laura Gonzales , L Nancy
Published: June 2021

Myogenic tone is an important regulator of vascular resistance and is increased in several models of hypertension. Ca2+ sparks can decrease vascular smooth muscle contraction and have been proposed to regulate myogenic tone. We previously reported that exposing rats to eucapnic intermittent hypoxia (E‐IH) to simulate sleep apnea causes hypertension and augments agonist‐induced vascular reactivity. We hypothesized that E‐IH increases myogenic reactivity and decreases Ca2+ spark frequency in rat mesenteric arteries. Small mesenteric arteries were dissected from rats exposed to E‐IH or air cycling (Sham) for 14 days. Intracellular Ca2+ concentration ([Ca2+]i) and inner diameter (i.d.) were recorded in pressurized arteries loaded with fura‐2 AM. Rapid intracellular Ca2+ events were recorded in arteries loaded with fluo‐4 AM. Elevating intraluminal pressure from 60 to 100 mmHg increased myogenic tone and global [Ca2+]i in arteries from E‐IH rats (13.20 ± 5.49 to 30.63 ± 5.56 % Ca2+‐free i.d. and 1.33 ± 0.11 to 1.56 ± 0.11 F340/F380) but not those from Sham rats (11.56 ± 2.95 to 13.97 ± 3.37 % Ca2+‐free i.d. and 1.30 ± 0.07 to 1.44 ± 0.08 F340/F380). The same pressure step tended to increase spark frequency in Sham but not E‐IH arteries. These results support our hypothesis that E‐IH augments the development of myogenic tone through increased global [Ca2+]i associated with decreased Ca2+ spark frequency.
Role of TRPV4 channels in agonist‐induced endothelial Ca2+ entry and vasodilation: Evidence from TRPV4‐deficient mice
Authors: Mendoza Suelhem A. , Gutterman David D. , Bubolz Aaron , Suzuki Makoto , Zhang David X.
Published: June 2021

Transient receptor potential vallinoid 4 (TRPV‐4), is a Ca2+‐permeant cation channel implicated in endothelial‐dependent vasorelaxation to mechanical stimuli but limited information exists for agonist‐induced responses. We hypothesized that TRPV‐4 is required for agonist‐induced vasodilation by regulating Ca2+ entry. Isometric tension was measured in isolated mesenteric arteries (MA) from wild type (WT) and TRPV‐4 knockout mice using a wire myograph. Dilation to acetylcholine (Ach, 10−9–10−5 M) was diminished in TRPV‐4−/− vs. WT MA (% max relaxation, 44±12 and 86±5, respectively n=4, P<0.05). L‐NAME, blocked Ach relaxation in TRPV‐4−/− and WT MA (11±4 and 35±11, respectively n=4, P<0.05). Responses to papaverine were similar in WT and TRPV‐4−/− MA. Intracellular calcium ([Ca2+]i) was assessed after intraluminal fura‐2 (fluorescence) in MA. Ach (10−5 M) increased endothelial [Ca2+]i in WT and TRPV‐4−/− MA (Δ[Ca2+]i, 49±4.6nM and 26±2.1nM, respectively n=4). The addition of 4α‐PDD, a TRPV4 opener, increased [Ca2+]i in WT but not in TRPV‐4−/− (Δ[Ca2+]i, 164±30nM and 15±1.3nM, respectively n=4). In endothelium of WT MA the increase in [Ca2+] was abolished by the TRPV‐4 blocker ruthenium red (Δ[Ca2+]i, RR, 19±1.2nM n=4). These data suggest an important role for the TRPV‐4 channels in vasodilation to Ach, and regulation of endothelial intracellular Ca2+in mice MA.
The tight junction proteins ZO‐1 and occludin undergo novel MLCK‐independent redistribution during TNF‐induced cell shedding
Authors: Marchiando Amanda M , Guan Yanfang , Hecht Aaron L. , Angus Elizabeth M. , Montrose Marshall H. , Turner Jerrold R. , Watson Alastair JM
Published: June 2021

Small intestinal epithelial cells are continuously shed from the villus at steady state. Small discontinuities, or epithelial gaps, occur during in vivo cell shedding. Our aims were to determine if tumor necrosis factor (TNF) increased rates of shedding and gap formation, the role of myosin light chain kinase (MLCK) in shedding, and whether the tight junction contributes to barrier maintenance at these sites.

Fluorescent‐tagged ZO‐1 or occludin expression in intestinal epithelia was driven by the villin promoter. Transgenic mice were injected with TNF (7.5μg, i.p.). Shedding was evaluated by video confocal microscopy. TNF increased shedding by 27‐fold (p<0.007), beginning 60 min after TNF, and caused reorganization of both ZO‐1 and occludin. Over the 10 min during which shedding occurred, both ZO‐1 and occludin migrated downward along lateral membranes and condensed beneath shedding nuclei to form a funnel that moved apically with the extruding cell. Extracellular probes (e.g. lucifer yellow) approached but did not cross the line of ZO‐1 and occludin, suggesting maintenance of the mucosal barrier. Tight junction remodelling was not altered via enzymatic MLCK inhibition or in MLCK knockout mice, suggesting that this process may not require MLCK.

We conclude that ZO‐1 and occludin undergo novel MLCK‐independent redistribution at sites of barrier maintenance during TNF‐induced cell shedding.

Supported by NIH.
Induced JunD in intestinal epithelial cells represses CDK4 transcription through dimerization with ATF‐2 following polyamine depletion
Authors: Xiao Lan , Rao Jaladanki N. , Zou Tongtong , Liu Lan , Chen Jie , Yu Tingxi , Turner Douglas J. , Wang Jian‐Ying
Published: June 2021

Intestinal epithelium is a rapidly self‐renewing tissue and its homeostasis is regulated by polyamines. Decreasing polyamines increases JunD inhibiting intestinal epithelial cell (IEC) renewal through CDK4 repression, but the mechanism by which JunD represses CDK4 remains elusive. ATF‐2 is an ATF/CREB transcription factor and its expression is regulated by polyamines. Here, we determined if JunD forms dimers with ATF‐2 repressing CDK4 transcription following polyamine depletion in IECs.

Methods: In vivo JunD binding was detected by immunoprecipitation assay (IP), while in vitro dimerization was examined by GST pull‐down assays. CDK4 transcription was measured by CDK4‐promoter luciferase reporter gene assays. ATF‐2 function was investigated by RNA interference.

Results: Induced JunD formed complexes with ATF‐2 as measured by IP, which was confirmed by GST pull‐down assays. Studies using deletion‐ and point‐mutations of ATF‐2 revealed that JunD/ATF‐2 dimer formation depended on the C‐terminal leucine zipper motif located between 380th to 413th amino acids in ATF‐2 sequence. JunD/ATF‐2 dimers were increased following polyamine depletion, which were associated with CDK4‐promoter inhibition. ATF‐2 silencing prevented CDK4 repression induced by JunD and enhanced IEC renewal.

Conclusions: Induced JunD represses CDK4 transcription through dimerization with ATF‐2 following polyamine depletion.
Hormonal Regulators of Muscle and Metabolism in Aging (HORMA) study: body composition and performance outcomes
Authors: Schroeder E Todd , Castaneda‐Sceppa Carmen , Binder Ellen , Wang Ying , Kawakubo Miwa , Yarasheski Kevin , Bhasin Shalender , Azen Stanley P , Sattler Fred R
Published: June 2021

We hypothesized that endogenous testosterone (T) and growth hormone (GH), regulate skeletal muscle and fat by different but complementary mechanisms and augmentation to youthful levels would enhance body composition and function in older, community dwelling men at risk for sarcopenia/frailty. HORMA is a placebo controlled, double‐blind, factorial study to assess T & GH dose levels in hyposomatotropic (IGF‐1 in lower tertile), low/hypogonadal (T 200–500 ng/dL) 65–90 yr old men for 16wks. 122 subjects were randomized to 6‐groups, transdermal T (5 or 10g/day) and rhGH (0, 3, or 5μg/kg/day). Table shows the primary results. In addition, appendicular lean, trunk fat, aerobic endurance time significantly improved (p<0.05) but muscle power, fatigability, VO2peak did not. T alone or with GH improved body composition and select measures of muscle function.
Susceptibility of the colon to psychological stress‐induced changes is increased by exposure to previous inflammation
Authors: Santiago Olga I , Cruz Myrella L , Cuevas Marielly , Appleyard Caroline B
Published: June 2021

Stress has been associated with exacerbation of symptoms in disorders such as Irritable Bowel Syndrome.

AIM:

To determine the effects of prior inflammation on subsequent stress‐induced changes in colonic cytokine and bacterial levels.

METHODS:

Four groups of female rats were used: normal (untreated); control‐WAS (water avoidance stress: for 3 hrs, during 3 consecutive days); control‐colitis, animals received trinitrobenzene sulfonic acid (TNBS 30mg in 50% ETOH ic) to induce an acute colitis which was allowed to heal for 2 weeks; and colitis‐WAS, the animals received TNBS as above, then WAS 2 weeks later. All animals were evaluated for weight change, macroscopic damage and histology. Colonic tissues were inoculated on blood, MacConkey or lactobacillus agar. Colonic tissue levels of IL‐10, IL‐4 and TGF were measured by ELISA.

RESULTS:

Stress increased weight loss in animals that previously had colitis compared to controls (p<0.05). These animals also showed increased macroscopic and microscopic damage, and mast cell numbers. Colitis‐WAS had the lowest levels of lactobacillus, but little change in total bacterial load. Colitis‐WAS had decreased levels of IL‐4 and IL‐10, and increased levels of TGF compared with control‐WAS.

CONCLUSION:

Previous inflammation increases the susceptibility of the colon to subsequent stress, with alterations in the microflora and immune response. F31 DK074378 & 2G12RR03050
Epidermal growth factor preserves intestinal integrity and decreases mortality in a murine model of Pseudomonas aeruginosa pneumonia
Authors: Clark Jessica Ann , Lawrance Christopher P. , Dunne William M. , Coopersmith Craig M.
Published: June 2021

Exogenous epidermal growth factor (EGF) attenuates intestinal injury and decreases mortality in a peritonitis model. However, the role of EGF in sepsis caused by extraabdominal infection is unknown.

We hypothesized that EGF preserves intestinal integrity and decreases mortality in P. aeruginosa pneumonia‐induced sepsis.

FVB/N mice were intratracheally injected with 40 μl of 0.6 McF P. aeruginosa and treated with or without EGF (150 ug/kg/d i.p.; n=9/group). Controls were injected with saline (n=5). All mice received antibiotics. At 24 hr, intestines were evaluated for apoptosis by caspase‐3 staining, proliferation by BrdU staining, and villus length. Survival studies were done in a separate cohort of mice (n=5–10/group).

Compared to shams, septic mice had increased intestinal apoptosis (36 ± 3 vs. 17 ± 3 cells/100 crypts; p<0.001) and decreased proliferation (502 ± 51 vs. 959 ± 43 cells/100 crypts; p<0.001), while EGF normalized both to sham levels (21 ± 1 vs. 17 ± 3 and 886 ± 64 vs. 959 ± 43 cells/100 crypts, respectively; p=ns). Septic mice had shorter villi compared to shams, and EGF treatment resulted in normalization to sham villi lengths (333 ± 15 vs. 415 ± 30 vs. 432 ± 19 μ m; p<0.01 and p=ns). At 7 days, septic EGF treated mice had a survival advantage compared to septic mice (92% vs. 50%; p=0.05).

EGF preserves intestinal integrity and improves survival in pneumonia‐induced sepsis; thus, the protective effects of EGF are not specific to the anatomical site or type of infection. EGF may be a novel therapeutic agent for the treatment of sepsis.
Deciphering the presenilin‐nicastrin interaction in the Alzheimer's disease related gamma‐secretase complex
Authors: Gentile Lisa Nancy , Preecs Sandlin , Chapin Anne , Rocha Monica , Franko Cloe
Published: June 2021

Alzheimer's disease (AD), the most common type of dementia, currently affects a rapidly increasing 5.1 million Americans. This ultimately fatal neurodegenerative disorder typically presents with mild short‐term memory loss which progresses to an inability to carry out regular daily functions and recognize loved ones. While the exact cause of AD is unknown, it is known that the characteristic build‐up of amyloid plaques around neurons plays an important role in the memory loss in AD patients. Amyloid plaques are composed of Abeta‐42 peptides produced by cleavage of the amyloid precursor protein by gamma‐secretase. Gamma‐secretase is a high molecular weight complex composed of presenilin 1 or presenilin 2, nicastrin, PEN‐2, and APH‐1a or APH‐1b. Research will be presented that shows that the C‐terminus of presenilin 1 (amino acids 300–467) binds to nicastrin (amino acids 312–369). Progress on the binding of mutant presenilins and nicastrins that lead to increased or decreased production of Abeta‐42 will also be presented. An increased awareness of the binding specifications of the presenilin 1 C‐terminus to nicastrin, necessary for the formation of the gamma‐secretase high molecular weight complex, will give insight into the production of Abeta‐42, and thus a greater understanding of ways in which its production may be blocked.
LPS inhibits endothelin‐1 (ET‐1)‐mediated endothelial nitric oxide synthase (eNOS) activation through the RhoA/Rho‐kinase (ROCK‐2) pathway in hepatic sinusoidal endothelial cells (SECs)
Authors: Kwok Willson , Lee Sang‐Ho , Korneszczuk Katarzyna , Culberson Cathy , Gray David L. , Clemens Mark G.
Published: June 2021

The complex interactions among ET‐1, its receptors, caveolin and the components of the caveolae in the regulation of nitric oxide (NO) production are important determinants of hepatic functions in sepsis and endotoxemia. However, the exact mechanism following stresses such as endotoxin is unknown. We hypothesize that endotoxin disrupts ET‐1 signaling via RhoA and its downstream effector ROCK‐2 in hepatic SECs. In SECs isolated from Sprague‐Dawley rat livers, Western blot analysis indicated that LPS increased RhoA and ROCK‐2 expressions. The inhibition of ROCK‐2 using a competitive inhibitor Y27632 increased eNOS activity (1.61 ± 0.12), as measured by the conversion of [3H]‐L‐Arginine to [3H]‐L‐citrulline. Dose and time response experiments indicated that preincubation of 10 μM Y27632 in SECs for 30 min was most effective in enhancing eNOS activity in control media. In LPS, eNOS activity was not significantly increased by the treatment of ET‐1 (1.35 ± 0.11) or Y27632 alone (1.35 ± 0.17). However, the combined treatment of Y27632 and ET‐1 rescued eNOS activity in LPS (1.49 ± 0.14) associated with an upregulation of eNOS‐Ser1177 phosphorylation. Images from immunofluorescence confocal microscopy indicated that Y27632 inhibition of ROCK‐2 did not alter ROCK‐2 localization. These results suggest that upregulation of RhoA/ROCK‐2 may contribute to the inhibition of eNOS following LPS. Supported by R01 DK38201‐19
Ferroportin residue C326 is critical for its interaction with hepcidin
Authors: Nemeth Elizabeta , Fernandes Augustine , Ganz Tomas
Published: June 2021

The hormone hepcidin binds to the Fe exporter Fpn and induces its internalization and degradation, decreasing Fe efflux from cells into plasma. Fpn mutations that confer resistance to hepcidin cause classical hemochromatosis with parenchymal Fe loading. We analyzed mutations C326S, C326T, N144D and N144T to address the mechanism of Fpn interaction with hepcidin. Human Fpn‐EGFP construct was subjected to site‐directed mutagenesis and analyzed in HEK293T cells. The Fpn mutants localized properly to the cell surface and exported Fe normally. However, they were either completely (C326S/T) or partially (N144D/T) resistant to hepcidin‐mediated internalization, and exported Fe even in the presence of hepcidin. 125I‐hepcidin binding was completely (C236S/T) or partially (N144D/T) abrogated.

C326 and C205 were reported to be cell surface thiols. A non‐permeable thiol‐dependent crosslinker linked hepcidin to Fpn but not in C326S/T mutants, and only partly in N144D/T mutants. Alkylation of Fpn‐expressing cells ablated 125I‐hepcidin binding. We next examined whether hepcidin binding depended on a transient SS‐bond formation between C205 and C326. C205S/T substitutions, however, only partially inhibited hepcidin uptake and crosslinking. Bacitracin had no effect on hepcidin uptake or Fpn internalization indicating that isomerisation of a C205‐C326 disulfide bond was not necessary. Thus the C326 thiol is located in or near the binding site of hepcidin and is essential for hepcidin binding and subsequent internalization of ferroportin. Our observations help explain the severity of hemochromatosis in patients with C326X mutations.
A direct effect of erythropoietin on hepcidin expression involving EPOR signaling
Authors: Pinto Jorge P , Ribeiro Sara , Pontes Helena , Thowfeequ Shifaan , Tosh David , Carvalho Félix , Porto Graça
Published: June 2021

Background/Aims: Hepcidin is the main iron regulatory hormone, controlling the systemic absorption and remobilization of iron from stores. In vivo studies showed that hepcidin is down‐regulated by erythropoiesis, anemia and hypoxia, which conforms to the need for iron input for erythrocyte production. The hormone erythropoietin (EPO) is the primary signal triggering erythropoiesis in anemia and hypoxia. Therefore, a direct involvement of erythropoietin in hepcidin regulation can be hypothesized. The aim of this study was to test the effect of EPO on hepcidin expression in vitro and to begin characterization of the mechanisms involved.

Methods: Fresh mouse hepatocytes and the HepG2 cell line were supplemented with physiological and supra‐physiological doses of EPO. Blocking of EPOR was performed with a specific antibody. Quantification of hepcidin and C/EBPA mRNA was performed by quantitative Real‐Time PCR. C/EBPA protein levels were quantified by Western blot hybridization.

Results: EPO regulates hepcidin expression in HepG2 cells and in mouse hepatocytes, in a dose‐dependent manner. This effect is mediated through EPOR signaling, since hepcidin expression is restored by EPOR‐blocking. A pattern of expression similar to hepcidin after EPO and α‐EPOR treatments was observed for CEBPA at mRNA and protein levels, suggesting the involvement of this transcription factor in the regulation of hepcidin by EPO.
Antenatal glucocorticoid at clinically relevant doses alter glucose tolerance in the adult offspring in sheep
Authors: Figueroa Jorge P. , Massmann Angela G , Zhang Jie , Rose James C
Published: June 2021

Exposure to glucocorticoids (GC) in the perinatal period is associated with alterations in glucose tolerance and hypertension in adult life. The aim of the present study was to study the effects of clinically relevant doses of GC on glucose tolerance and adipose tissue leptin expression.

Pregnant sheep were treated with two IM doses of betamethasone (BM, 0.17 mg/kg) or vehicle 24‐hours apart at 80 days gestational age (term 145) and allowed to deliver at term. Adult offspring (12–18 months of age) of both sexes were chronically instrumented under general anesthesia to place intravascular catheters. Insulin sensitivity was evaluated both by iv glucose tolerance test (IVGTT) and euglycemic clamp (HEC) techniques. Leptin mRNA expression was determined by RPA in perirenal, omental and subcutaneous fat. Data Mean±SEM were analyzed by two way ANOVA and/or t test.

A significant time and treatment (BM) effect was observed in plasma glucose during the IVGTT. BM also was associated with higher insulin plasma levels, but did not reach statistical significance. Leptin expression was significantly higher in perirenal but not omental adipose tissue of BM exposed sheep [0.28±0.1 vs 0.88±0.2;0.60±0.2 vs 1.0±0.2]. Exposure to a single course of GC at 0.55 gestation has longterm effects on glucose metabolism regulation. BM‐exposed sheep exhibit alterations in glucose tolerance, hyperinsulinemia and insulin resistance. HL 68728.
Gene expression profiling of the RAS in myoblasts following differentiation and mechanical stretch
Authors: Johnston Adam P , Baker Jeff , Parise Gianni
Published: June 2021

Recent evidence, using animal models, suggests that angiotensin II can positively influence skeletal muscle hypertrophy. We conducted a genetic profile of RAS family members during C2C12 myoblast proliferation and differentiation. We also examined RAS family member gene expression following 4h of mechanical stretch. Both angiotensin II receptors (AT1 and AT2) were expressed in proliferating and differentiating myobasts and were significantly upregulated at day 1 of differentiation, while the AT2 receptor demonstrated a persistent upregulation at both day 1 and day 3 as compared to proliferation. Interestingly, angiotensiogen was transiently downregulated upon exposure to differentiation media but was significantly upregulated at day 7 of differentiation. Furthermore, angiotensin converting enzyme showed weak expression during proliferation but increased significantly upon differentiation across all time points. Immediately following mechanical stretch we observed a reduction in AT1 and AT2 gene expression. In addition we observed an increase in AT1 expression 4h post stretch. These results suggest that RAS family members may play a role in regulating myoblast differentiation. Furthermore, it appears that RAS family members are responsive to stretch‐induced stress.
Arachidonic acid (AA) inhibits basolateral K channels in the cortical collecting duct (CCD) via cytochrome P450 (CYP) epoxygenase‐dependent metabolic pathways
Authors: Wang Zhijian , Wei Yuan , Wang Wenhui
Published: June 2021

We used the patch‐clamp technique to study the effect of AA on basolateral 18 pS K channels in principal cell of the CCD of the rat kidney. Application of AA inhibited the 18 pS K channels. The effect of AA on the 18 pS K channel was specific because application of 11,14,17‐eicosatrienoic acid had no effect. Also, the inhibitory effect of AA on the 18 pS K channels was abolished by blocking CYP epoxygenase with MS‐PPOH but was not affected by DDMS, an inhibitor of CYP monoxygenase. The notion that the inhibitory effect of AA was mediated by CYP epoxygenase‐dependent metabolites was also supported by the observation that application of 100 nM 11,12‐epoxyeicosatrienoic acid (EET) mimicked the effect of AA and inhibited the basolateral 18 pS K channels. In contrast, addition of either 8,9‐, or 14,15‐EET failed to inhibit the 18 pS K channels. Moreover, application of 11,12‐EET was still able to inhibit the 18 pS K channels in the presence of MS‐PPOH. This suggests that 11,12‐EET is a mediator for the AA‐induced inhibition of the 18 pS K channels. We conclude that AA inhibits basolateral 18 pS K channels in the CCD and that the effect of AA is mediated by a CYP epoxygenase‐dependent metabolite, 11,12‐EET.
A natural plant derived‐dihydroisosteviol prevents cholera toxin‐induced intestinal fluid secretion
Authors: Chatsudthipong Varanuj , Pariwat Prapapimon , Homvisasevongsa Sureeporn , Muanprasat Chatchai
Published: June 2021

Stevioside and its major metabolite, steviol, have been reported to affect ion transport in many types of tissues, such as kidney, pancreas and intestine. The effect of stevioside, steviol and its analogs on intestinal Cl− secretion was investigated in a human T84 epithelial cell line. Short circuit current measurements showed that steviol, and analogs, isosteviol, dihydroisosteviol and isosteviol 16‐oxime, inhibited in a dose‐dependent manner forskolin‐induced Cl− secretion with IC50 of 101, 100, 9.6 and 50 μM, respectively, whereas the parent compound stevioside had no effect. Apical Cl− current measurement indicated that dihydroisosteviol targeted CFTR. The inhibitory action of dihydroisosteviol was reversible and was not associated with changes in intracellular cAMP level. In addition, dihydroisosteviol did not affect calcium‐activated chloride secretion and T84 cell viability. In vivo studies using a mouse closed loop model of cholera toxin‐induced intestinal fluid secretion showed that intraluminal injection of 50 μM dihydroisosteviol reduced intestinal fluid secretion by 89% without altering fluid absorption. These results indicate that dihydroisosteviol and similar compounds could be a new class of CFTR inhibitors useful for further development as antidiarrheal agents.
Valproate and Nonsteroidal Anti‐Inflammatory Drugs Reduces the Activity of Electroneutral (Smtn/Slc5a12) and Electrogenic (SMCTe/Slc5a8) Na+/Monocarboxylate Transporters (SMCT).
Authors: Plata Consuelo , Juárez Maria Eugenia , Vázquez Norma , Gamba Gerardo , Romero Michael F
Published: June 2021

The electroneutral (zSMCTn/Slc5a12) and electrogenic (zSMCTe/Slc5a8) Na+/monocarboxylate (MC−) transporters of zebrafish, move short chain fatty acids, ketone‐bodies, lactate, pyruvate and nicotinate but with clear differences in Na+: MC− stoichiometry and affinity. The affinity of zSMCTn for pyruvate, lactate and nicotinate is lower than zSMCTe (~6, ~10 and ~40 times respectively). It is known that valproate and nonsteroidal anti‐inflammatory drugs, such as ibuprofen, valproate reduce the activity of mammalian SMCTe, but this effect has not been characterized for SMCTn. Using 22Na+ uptake in Xenopus oocytes injected with either zSMCT's or hSMCT cRNA, we evaluated the kinetics of Na+ propionate cotransport by SMCT's (hSMCTe, zSMCTe, zSMCTn). We also determined cotransport inhibition by ibuprofen, ketoprofen, naproxen and valproate of these oocytes in presence or absence of propionate. Our results indicate that zSMCTn (Km =830±110μM) has only 3 times less affinity for propionate than zSMCTe (Km =240±50μM) and hSMCTe (Km =200±20μM). Ibuprofen, ketoprofen, naproxen and valproate cause a significant reduction in 22Na+ uptake for zSMCTe, hSMCTe as well as zSMCTn in presence of propionate. Ibuprofen inhibition is more potent for zSMCTn (Ki~200μM) than zSMCTe (Ki~600μM) and hSMCTe (Ki~800μM). These functional and inhibitory differences may help define the binding site for MC− and Na+ of SMCT proteins.
Insulin resistance, secretion and breakdown are increased 6 months following severe burn injury
Authors: Cree Melanie G , Fram Ricki Y , Barr David , Chinkes David , Wolfe Robert R , Herndon David N
Published: June 2021

Insulin resistance following acute burns is well described, however, it is unknown how long this condition persists. Our objective was to measure the duration of insulin resistance following recovery from the acute burn injury. We performed a prospective cross‐sectional study with a 2 hr oral glucose tolerance test in 46 severely burned children at 6, 9 or 12 months (mo) following injury. Glucose uptake and insulin secretion were assessed, and compared to those previously published in healthy children. At 6 mo post‐burn, the 2 hr glucose was (P<0.001) greater than controls, and the area under the curve (AUC) of glucose was higher compared to 12 mo and to healthy children (P=0.027 and P<0.001, respectively). The 9 mo AUC glucose was higher than controls (P<0.01). The 6 mo 2 hour insulin in was higher than controls, as was the AUC of insulin in all time points post‐burn. The AUC of C‐peptide was greater at 6 mo post‐injury compared to 9 and 12 mo (P<0.01 for both). In conclusion, increased 2 hr and AUC glucose and insulin indicate that glucose metabolism is still altered at 6 and 9 mo post‐injury, coinciding with previously documented defects in bone and muscle metabolism at these times.
Aseptic externalized catheter placement and patency in cage confined and exercising yucatan miniature swine
Authors: Fleenor Bradley Stephen , Ivey Jan , Bowles Douglas K.
Published: June 2021

Chronic venous catheters in swine often malfunction and can lead to infection. Because of the similarities in human coronary anatomy the use of swine in biomedical research is an excellent model for studying cardiovascular disease. Here we describe the aseptic placement of externalized jugular catheters in 14 Yucatan miniature swine which remained patent (86%) and infection free (93%) for 28 days. Furthermore, 8 of the 14 swine underwent ∼ 90 minutes of treadmill exercise 20 of 28 days whereas the remaining 6 were cage confined. Following a small paratrachel incision, a custom designed trocar was used to subcutaneously tunnel a tygon catheter from the ventral to the midline of the dorsal neck. The proximal catheter was inserted into the jugular vein and secured 2 mm cranial to the heart within the vena cava. The distal catheter was exposed dorsally (1.5 cm) with a 17F stub adaptor and injection cap which allowed for drug injections as well as catheter flushing (3 times a week) and lock (1ml of 1000u/ml heparin). One animal that did develop an infection presented with signs late but still had a functional catheter. Our results demonstrate that external catheters can be highly functional and infection free in sedentary and exercised swine.

*Authors contributed equally

Supported by: NIH HL 52490
O‐GlcNAcylation increases vascular reactivity in rat aorta
Authors: Lima Victor Vitorino , Giachini Fernanda R. C. , Carneiro Fernando Silva , Webb R. Clinton , Tostes Rita C.
Published: June 2021

Introduction: O‐Linked N‐acetylglucosaminylation (O‐GlcNAcylation) plays an important role in many aspects of protein function. Whereas elevated O‐GlcNAc levels contribute to diabetes related end‐organ damage, O‐GlcNAcylation is also physiologically important.

Aim: Because proteins that play a role in vascular tone regulation can be O‐GlcNAcylated, we hypothesized that O‐GlcNAcylation increases vascular reactivity to contractile stimuli.

Methods: Aortas from Sprague‐Dawley male rats were incubated for 24 h with vehicle, PugNAc (O‐GlcNAcase inhibitor, 100μM), azaserine (GFAT inhibitor, 20μM), high glucose (25mM) or manitol (25mM).

Results: PugNAc incubation, but not azaserine, manitol or high glucose, significantly increased O‐GlcNAc‐proteins (Fig A), as determined by Western blot. PugNAc also increased phenylephrine and KCl contractions, which was not observed in the presence of L‐NAME. PugNAc decreased acetylcholine (Fig B), but not sodium nitroprusside, relaxation. Incubation with azaserine, high glucose or manitol did not change vascular reactivity. PugNAc incubation augmented e‐NOS levels, but decreased e‐NOS1177 phosphorylation.

Conclusions: Vascular O‐GlcNAcylation modulates vascular reactivity, possibly due to its effects on e‐NOS expression and activity, reinforcing the idea that O‐GlcNAcylation is important in physiological conditions.

Support: NIH / CAPES
Enhanced expression of pluripotency gene Oct‐4 in pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension
Authors: Yao Weijuan , Firth Amy L. , Ogawa Aiko , Yuan Jason X.‐J.
Published: June 2021

Pulmonary vascular remodeling is a major cause for the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). This study aimed at testing the hypothesis that the vascular medial hypertrophy may partially result from the de‐differentiation of pulmonary artery smooth muscle cells (PASMCs). RT‐PCR was performed to determine transcripts of pluripotency genes (Oct‐4/Nanog) and progenitor cell markers (CD34/Flk‐1) in PASMCs from normal subjects (n=3) and IPAH patients (n=3). The mRNA level of Oct‐4 in IPAH PASMCs was significantly higher than in normal PASMC, whereas Nanog was not detectable in IPAH and normal cells. The mRNA level of CD34/Flk‐1 was very low and no difference was found between IPAH and normal cells. Oct‐4 is a transcription factor highly expressed in undifferentiated cells (e.g., embryonic stem cells and multipotent adult progenitor cells). The upregulation of Oct‐4 indicates that PASMCs from IPAH patients are in the de‐differentiated state, and the transition from a differentiated (contractile and synthetic) phenotype to a de‐differentiated (proliferative) phenotype may serve as an important cause for vascular medial hypertrophy in IPAH.

Founding source: NIH grants (HL064945, HL054043, HL066012)
Tempol treatment ameliorates pulmonary hypertension in the TG (mREN2) 27 rat
Authors: DeMarco Vincent Gerard , Habibi Javad , Whaley‐Connell Adam T. , Andresen Bradley T. , Bosanquet James Percival , Delcour Kimberly S. , Schneider Rebecca Irene , Hayden Melvin R. , Sowers James R. , Dellsperger Kevin C.
Published: June 2021

Background: We recently reported that Ren‐2 rats, which express the mouse renin gene in extra‐renal tissues leading to local Ang II formation and hypertension, exhibit pulmonary hypertension (PH) and remodeling small pulmonary arteries. Here we test the hypothesis that PH and pulmonary vascular remodeling in the Ren‐2 rat is modulated by intrapulmonary oxidative stress.

Methods: 6 week old Ren‐2 and Sprague Dawley (SD) rats were treated with tempol (superoxide scavenger) or vehicle for 3 weeks. Right ventricular systolic pressure (RVSP), wall thickness of small pulmonary arteries (PA), intrapulmonary NADPH oxidase activity and reactive oxygen species (ROS) were compared.

Results: RVSP was higher in Ren2 compared to SD (61±7 mmHg vs 36±1; p<0.01); RVSP of tempol treated Ren2 (43±6 mmHg) was not different from untreated SD. Compared to SD, medial surface area of PA was 38% greater (p<0.001) and luminal surface area was 54% less (p<0.001) in Ren2; tempol treatment prevented these changes (p<0.05). ROS and NADPH oxidase activity were elevated in Ren2 lungs compared to SD (p<0.05) and tempol treatment prevented these increases (p<0.05).

Conclusions: Ren2 rats exhibit intrapulmonary oxidative stress, medial thickening of PA, and PH, and tempol treatment ameliorates these changes. This study supports a role for enhanced tissue RAS/oxidative stress as factors in development of PH and pulmonary vascular remodeling.
Increased cavernosal relaxation in type 2 diabetic Goto‐Kakizaki rats
Authors: Carneiro Fernando S. , Giachini Fernanda R. C. , Carneiro Zidonia N. , Lima Victor V. , Ergul Adviye , Tostes Rita C. , Webb R. C.
Published: June 2021

Introduction: Diabetes mellitus (DM) is an important risk factor for erectile dysfunction (ED). Commonly used experimental models of diabetes include the chemically‐induced streptozotocin model of type 1 diabetes, which displays highly elevated glucose levels, or obesity‐induced models such as Zucker rats and db/db mice.

Aim: In this study we evaluated cavernosal reactivity in Goto‐Kakizaki (GK) rats, which are not obese and display moderate changes in blood glucose, insulin resistance and blood pressure. This model is particularly relevant to ED studies since the great majority of patients with prediabetes or type 2 diabetes display mild defects in glucose‐stimulated insulin secretion, insulin resistance, hyperinsulinemia and hyperglycemia.

Methods and Results: Male GK and Wistar control (C) rats were used at 18 weeks of age (n=5 and 7, respectively). Sympathetic‐mediated contractile responses (induced by electrical field stimulation (EFS), 50V, 1 ms pulse, 1–64 Hz) were attenuated in cavernosal strips from GK rats (C: 6.3±0.7 vs GK: 2.5±0.4, mN). Similarly, cavernosum from GK rats showed decreased contractions to phenylephrine (C: 7.0±0.4 vs GK: 1.9±0.2, mN) and KCl (C: 4.6±0.3 vs GK: 1.4±0.2, mN). Non‐adrenergic‐non‐cholinergic (NANC)‐induced relaxation was increased in cavernosal strips from GK rats (Figure 1), but relaxation to sodium nitroprusside was not.

Conclusion: In contrast to other models of diabetes, GK rats exhibit changes in cavernosal reactivity that would facilitate erectile responses. This may be due to compensatory mechanisms activated at this stage of diabetes.