bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA‐DEPENDENT PROTEIN KINASE IS CRITICAL FOR VCAM‐1 EXPRESSION UPON TNF TREATMENT THROUGH PHOSPHORYLATION OF p50 NF‐kappa B: A CRITICAL INVOLVEMENT IN VASCULAR AND LUNG INFLAMMATION
Authors: Ju Jihang , Naura Amarjit , Errami Youssef , Kim Hogyoung , Kim Jong , Beg Amer A. , Giardina Charles , Boulares Hamid
Published: June 2021

NF‐κB is critical for vascular cell adhesion molecule 1 (VCAM‐1) expression during vascular inflammation. We show that DNA‐dependent protein kinase (DNA‐PK) is required for VCAM‐1 expression in response to TNF‐α in an established cell line and in primary endothelial cells. DNA‐PK deficiency, achieved by DNA‐PKcs heterozygosity, reduced VCAM‐1 expression in the aorta in an animal model of septic shock and blocked airway neutrophilia in an animal model of acute lung injury. Interestingly, such effect unravels a novel function for DNA‐PK independent of its role in immune competence. Phosphorylation and nuclear translocation of p65 NF‐κB (p65) was insufficient for VCAM‐1 expression in response to TNF‐α. The requirement for p50 NF‐κB (p50) was associated with its interaction with and phosphorylation by DNA‐PK, which appears to be dominant over the requirement for p65 activation. Additionally, DNA‐PK activity appeared to increase the κB‐site binding ability of p50/p50 and p50/p65 dimers to VCAM‐1 promoter. Serine 20 in p50 was found to be phosphorylated by DNA‐PK. Re‐establishing wild‐type p50, but not its S20/A mutant, in p50‐knockout cells reversed VCAM‐1 expression. Together, these results elucidate a novel mechanism for the regulation of VCAM‐1 expression by DNA‐PK through phosphorylation of p50 and its critical involvement in vascular and lung inflammation.
NaPiIIb in rat enterocytes interacts with EBP50 and Shank2
Authors: Rachelson Joanna , Cranston DeeAnn , Dobrinskikh Evgenia , Levi Moshe , Doctor R. Brian
Published: June 2021

Sodium‐phosphate co‐transporter type IIb (NaPiIIb) is a major phosphate transporter in enterocytes of the small intestine. Normally concentrated within the apical microvilli, little is known about the molecular mechanisms that moderate the distribution and activity of NaPiIIb. Sequence analysis of NaPiIIb reveals a putative PDZ binding motif on its C‐terminal tail. Western blotting indicates NaPiIIb is clearly expressed in the duodenum and jejunum while potential PDZ protein binding partners, including EBP50 and Shank2, are expressed in the duodenum, jejunum and ileum. Like NaPiIIb, EBP50 and Shank2 are concentrated within the apical microvilli of duodenal enterocytes. Enterocytes that showed appreciable levels of NaPiIIb within the cell interior had increased levels of Shank2 also within the cell interior. Studies in native enterocytes found NaPiIIb co‐precipitated with EBP50 and Shank2. Studies of recombinant proteins in HEK cells found GFP‐NaPiIIb co‐precipitated with both Flag‐EBP50 and Flag‐Shank2. These interactions were markedly diminished when the last four amino acids, corresponding to the PDZ binding motif, were truncated from the C‐terminus of NaPiIIb. These findings indicate PDZ domain proteins bind to NaPiIIb and are positioned to moderate the distribution and activity of NaPiIIb.
Acute activation of the renal betaine/GABA transporter by low extracellular calcium
Authors: Parikh Nehal R , Vaughn Cherissa L , Kempson Stephen A
Published: June 2021

The betaine/GABA transporter (BGT1) is important for osmoprotection in kidney medullary cells. We previously reported an acute (30 min) increase in extracellular Ca caused dose‐dependent inhibition of BGT1 in renal MDCK cells (AJP Renal 291:F305‐12, 2006). To determine if extracellular Ca might be a local regulator of BGT1, we tested the response to low Ca serum‐free growth medium (LCM, 0.09 mM Ca2+). Na+/GABA cotransport in MDCK monolayers was activated within 30 min after transfer from standard culture medium to LCM. Activation was significant after 60–90 min (n=3–5) and was independent of medium osmolarity. Peak transport was increased 50% in isotonic LCM and 100% in hypertonic (500 mOsm) LCM over controls. Hypertonic LCM also activated Na+/betaine cotransport by 400% within 60 min (n=3). The system A amino acid transporter showed a similar 4‐fold activation in hypertonic LCM. Perfusion of Fura‐ 2‐loaded MDCK cells with LCM decreased intracellular Ca2+ by 31% (n=22) within 6–7 min. Confocal imaging of ZO‐1 protein showed a marked change in distribution within 45 min exposure to LCM, consistent with disruption of tight junctions. Trafficking pathways may be disrupted by loss of tight junctions and cell polarity, and activation of BGT1 and system A transport by LCM could be due to increased membrane insertion or decreased retrieval.
Isolation of the TRPV4 Osmoreceptor from the Avian Lower Gastrointestinal Tract
Authors: Warner Paige C , Braun Eldon J
Published: June 2021

Avian osmoregulation differs from mammals in that not only do the kidneys regulate water and electrolyte levels but the lower gastrointestinal tract as well. Urine from the kidneys is conveyed to the cloaca where a reverse peristalsis brings the urine in contact with epithelial tissues of the colon and digestive ceca where its composition can be further modified. Brummermann and Braun (AJP, 268, 1995) have shown not only do changes in osmolarity within the lumen of the cloaca have a direct effect on reverse peristalsis, but also that this occurs independent of central osmoreceptors, suggesting local regulation of the reverse peristalsis. It is believed that the sensor of osmolarity is a member of the vanilloid‐receptor subfamily of transient receptor potential channels (TRPV4). TRPV4 has been shown to respond to osmotic stimuli in a variety of mammalian tissues. Cloacal and colonic tissues were harvested from mourning doves and homogenized for analysis using Gel Electrophoresis, Western blotting, and Immunohistochemistry. TRPV4 was localized to the area where the ureters enter the cloaca, but not found in other regions of the cloaca. In addition, TRPV4 was not found in the colon suggesting that the osmotic potential of the urine is monitored as it enters the cloaca. We used rat renal tissue where TRPV4 has been previously shown, as a control and clearly demonstrated the presence of TRPV4 in the medullary region of the kidney.
Estimating Water Loss During Hibernation in the American Black Bear (Ursus americanus)
Authors: Howard Stephen Andrew , Rothstein Krista , Laske Timothy G , Garshelis David L , Iaizzo Paul A
Published: June 2021

The American black bear can hibernate for up to 6 months. During this period they do not eat, drink, urinate, or defecate: they elicit minimal movements and typically do not leave their dens. Still, they lose minimal skeletal muscle mass and strength, and do not suffer any adverse consequences of either starvation or dehydration. In previous reports bears have been shown to maintain normal blood compositions, suggesting that they sustain homeostasis of their blood and other bodily fluids. To maintain this water balance without any fluid intake, bears must minimize water loss throughout their bodies, including their respiratory systems.

Radio‐collared wild black bears in northern Minnesota were tracked to their dens during early or late hibernation. Bears were anesthetized, after which the relative amount of water vapor expired was analyzed using a hygrometer/mask system. We designed a respiratory mask to fit on the muzzle of the bear to collect expired air while still allowing them to breathe normally. From these data we estimated the amount of water vapor lost by bears during hibernation and were able to estimate that a 200–250 lbs bear would lose only 2.3L of water in 5 months of hibernation. This ability to conserve water during respiration is required to sustain long periods without intake through diet or drinking. Funding by NIH training grant 5T32AR007612 and Medtronic professorship funding.
Search For RAGE In Avian Vasculature
Authors: Eythrib Farid J , Braun Eldon J
Published: June 2021

Advanced glycation end‐products (AGEs) are the products of non‐enzymatic reactions between serum albumin and glucose that occur as the first stage of glycation. Receptor for advanced glycation end‐products (RAGE) is a transmembrane protein which binds AGEs. RAGE was first characterized as a member of an immunoglobulin protein family (Neeper et al. 1992). When AGE's bind to RAGE, an inflammatory response is initiated. Although this is most likely a natural immune response that results from high levels of AGEs in the blood, i. e. diabetes, the inflammation caused by activation of RAGE can cause damage to the vasculature by altering the microenvironment of the basal membrane. This creates what can be described as “leaky” vasculature (edema), which can be detrimental to patients suffering from diabetes. The mourning dove, which has a blood glucose level 4–5 times higher than that of a fasting diabetic human, does not suffer from these RAGE‐related symptoms. It was hypothesized that birds may not express the RAGE protein; however, early SDS‐Page electrophoresis and Western Blots have identified the likely presence of RAGE in mourning dove vasculature when treated with a RAGE‐specific antibody derived from rabbits. Western blot techniques will be used to more clearly identify the putative RAGE protein from avian tissue as well as immunocytochemistry will be used to localize at what level RAGE exist in the vascular tree.
In‐vivo quantification of 3D muscle architecture in Triceps Surae muscle
Authors: Rana Manku , Wakeling James M.
Published: June 2021

Fascicle orientation is an important architectural parameter affecting the mechanical function of skeletal muscle. Most previous studies on fascicle architecture have been in 2D and the importance of 3rd dimension is not much explored. The 3D orientation of the whole muscle may be regionalized and is not uniform across the whole muscle. We have quantified the muscle fascicle orientation in soleus and gastrocnemii muscles in six male subjects for three contraction states (0%, 30% and 60% of MCV) and four ankle angles ( −15°, 0°, 15° and 30° of planter flexion) using B‐mode ultrasound and optical tracking systems. Images were obtained from multiple scans of the muscles with scan times less than two minutes. The images were analyzed for 3D fascicle orientation represented in a muscle‐based spherical co‐ordinate system in terms of (pennation) angle β with the long axis of the muscle and angle ϕ made by the projection of the fascicle in the transverse plane of the muscle. Both β and ϕ were regionalized across the muscles and changed with the different ankle angles and contraction states (p<0.01). Muscle fascicles are arranged in sheets and these sheets can bulge when a muscle contracts and hence the fascicle may no longer lie in a 2D plane. We suggest that the fascicle arrangement changes in response to intramuscular pressure, and these changes alter the mechanical output. This project is funded by NSERC.
Regulation of GLUT1‐mediated glucose and dehydroascorbic acid (DHA) transport
Authors: Andrisse Stan , Patel Gaytri D. , Chen Joseph E. , Webber Andrea M. , Spears Larry D. , Fisher Jonathan S.
Published: June 2021

The GLUT1 PDZ‐binding motif that plays a role in GLUT1 localization contains a reported phosphorylation site, S490, for ataxia telangiectasia mutated (ATM). The objective of this study is to determine whether ATM and/or modification of GLUT1 S490 affect GLUT1 localization, trafficking, and transport activity. Mouse extensor digitorum longus (EDL) muscles were excised from wild type and ATM −/− mice and then subjected to a DHA transport assay. ATM inhibition by KU55933 or CP466722 or ATM dysfunction (ATM−/−) in EDL resulted in reduced DHA transport (~30–65%, p<0.05). Also, L6 myoblasts were transfected with FLAG tagged GLUT1 (FLAG‐GLUT1) or FLAG‐GLUT1 S490A and S490D site mutants. ATM inhibition significantly diminished cell surface FLAG‐GLUT1 (~20–80%, p<0.05) and glucose transport in L6 myotubes (~50%, p<0.05) with a concomitant reduction in GLUT1 externalization (~20%, p<0.05). The S490D mutation increased cell surface GLUT1 (~35%, p<0.05) as well as GLUT1‐mediated glucose transport (~2‐fold, p<0.05), while S490A had opposite effects. These results provide evidence that ATM and GLUT1 S490 mutations affect cell surface GLUT1 and GLUT1‐mediated transport. Supported by USPHS award 1R15DK091904.
Inactivation of STAC3 causes perinatal death and defects in skeletal muscle in mice
Authors: Reinholt Brad Michael , Ge Xiaomei , Park Sungkwon , Gerrard David , Jiang Honglin
Published: June 2021

The Src homology 3 domain (SH3) and cysteine rich domain (C1) 3 (STAC3) gene is a novel gene copiously expressed in skeletal muscle. Our objective was to determine the role of STAC3 in development. We used a knockout approach to evaluate the phenotypic effects of STAC3 gene deletion on development in mice. Homozygous STAC3 null (KO) mice appeared cyanotic and died perinatally, but possessed otherwise normal organs based on histological evaluation. The primary phenotypes displayed in KO mice were reduced body weights and increased percentage of myotubes with centrally located nuclei when compared to heterozygous (HET) and wild type (WT) littermates. At embryonic day 18.5 (E18.5) KO mice displayed a 12.7% reduction in weight (P<0.001). However, at E15.5 weights and morphology of mice were similar. At birth (P0) and at E17.5, KO mice had 59% and 24% more myotubes with centrally located nuclei, respectively, than HET or WT littermates (P<0.001). Overall, these data show STAC3 is necessary for development of viable offspring and suggest STAC3 plays a crucial role in embryonic development where its primary phenotype is exhibited in skeletal muscle. Additional work is necessary to identify the underlying mechanism of STAC3 in fetal development.
Norepinephrine in vivo restores the low phosphorylation levels of Akt and attenuates the high expression of atrophy‐ and autophagy‐related genes in skeletal muscles of fasted mice
Authors: Silveira Wilian Assis , Gonçalves Dawit A. P. , Graça Flávia A. , Zanon Neusa M. , Kettelhut Isis C. , Navegantes Luiz C. C.
Published: June 2021

It is well known that catecholamines inhibit skeletal muscle protein degradation, but the molecular underlying mechanism remains unclear. This study was undertaken to investigate the in vitro and in vivo effects of norepinephrine (NOR) on regulating the ubiquitin‐proteasome (UPS) and autophagic/lysosomal systems. The proteolytic activities were determined by measuring the tyrosine release in rat isolated muscles. The gene expression and levels of Akt phosphorylation were examined by RT‐qPCR and Western blotting analysis, respectively. The addition of NOR to the incubation medium of extensor digitorum longus (EDL) muscles from fed rats reduced the basal UPS activity (21%), the expression of atrophy‐related genes, Atrogin‐1 (64%) and MuRF‐1 (43%), and autophagy‐related genes, LC3b (23%) and GABARAPl1 (23%). In C57/BL6 mice, 1‐d fasting reduced the phosphorylation levels of Akt‐Ser473 and up‐regulated the expression of atrophy‐ and autophagy‐related genes in tibialis muscles. One single injection of NOR reestablished the low levels of p‐Akt and attenuated the high expression of atrogin‐1 (63%), MuRF‐1 (60%) and GABARAPl1 (55%) in fasted mice. These data indicate that NOR attenuates the fasting‐induced expression of atrophy‐ and autophagy‐related genes in skeletal muscles of mice, probably involving activation of Akt signaling pathway. Financial support: FAPESP (10/11083‐6; 08/06694‐6).
Absolute quantification of muscle glycogen content in patients with glycogen storage disease by 13C NMR spectroscopy at 7 Tesla
Authors: Heinicke Katja , Dimitrov Ivan , Cheshkov Sergey , Malloy Craig R. , Haller Ronald G.
Published: June 2021

Objective

Evaluate the invivo muscle glycogen content in patients with glycogen storage disease by performing noninvasive 13C NMR spectroscopy in ultra‐high fields with the advantages of improved signal‐to‐noise ratio and spectral resolution.

Methods

Muscle glycogen content was measured in 3 patients with McArdle disease (MD), 1 phosphofructokinase deficient (PFKD) patient, and 4 healthy controls (C). 13C spectra were acquired on a whole‐body 7T scanner (Philips Medical) using a partial volume human calf coil operating in quadrature for 1H and 13C. The coil had a small vial of acetate fixed inside, to serve as an external reference for quantification. For absolute quantification, a 1L cylindrical phantom was prepared with 100 mM glycogen, and 40 mM creatine.

Results

Muscle glycogen concentration was increased in MD (184.7±14.9 mM) and PFKD (196.0 mM) compared with C (73.1±13.0 mM). Creatine concentration and glycogen/creatine ratio respectively were 82.2±21.9 mM and 2.3±0.6 in MD, 120.5 mM and 1.6 in PFKD, 74.5±8.3 mM and 1.0±0.3 in C. T1 values, the strongly field dependent relaxation times, were established at 7T for glycogen 3.1±1.4 s and creatine 8.8±5.7 s in MD, 0.8±0.4 s and 2.0±1.4 s in C, respectively.

Conclusions

Muscle glycogen concentration was 2.5‐fold increased in patients with glycogen storage disease. Noninvasive ultra‐high filed 13C NMR spectroscopy may provide a valuable tool to study glycogen storage in progression of the pathology and efficacy of treatment.

Study supported by Muscular Dystrophy Association and Giant Tiger Foundation.
Functional decline, remodeling, and compensation in the respiratory system of a canine model of Duchenne muscular dystrophy
Authors: Mead Andrew , Malik Alock , Petrov Mihail , Childers Martin , Bogan Janet , Seidner Gabriel , Kornegay Joe , Stedman Hansell
Published: June 2021

Patients with Duchenne muscular dystrophy (DMD) experience a progressive loss of pulmonary performance and eventual insufficiency. The clinical course is well known, but the inability of the DMD patient to raise his metabolic rate has prevented respiratory reserve from being measured directly. The golden retriever muscular dystrophy (GRMD) dog model of DMD exhibits many of the hallmarks of the human disease, including respiratory muscle dysfunction. We used the respiratory stimulant doxapram in anesthetized dogs to simulate the effects of increased metabolic demand on respiratory drive while measuring multiple respiratory parameters. We found indicators of reduced reserve – reduced maximum tidal volume and ventilatory rate – and altered chest wall kinematics. GRMD diaphragms were severely hypertrophic and foreshortened. The abdominal contribution to inspired volume, an indicator of diaphragm involvement, was severely reduced in GRMD dogs. We propose that this remodeling of the diaphragm is adaptive, and allows the GRMD dog to employ accessory respiratory muscles to store elastic energy during expiration, aiding inspiratory pressure and flow and delaying the onset of respiratory insufficiency.
Regulation of the Circadian Clock in Human Blood Vessels
Authors: Wang Yusi , Fulton David
Published: June 2021

Approximately 5–10% of the mammalian genome oscillates with a circadian rhythm. Changes in gene expression are orchestrated by core circadian clock genes including Bmal1 and clock/NPAS2 which increase expression by target genes by binding to promoter E‐boxes. In contrast, Per and Cry genes reciprocally inhibit the activity of Bmal1:Clock/NPAS2. Recent studies revealed that expression of circadian clock genes, Bmal1, Clock, Per and Cry oscillate in murine arteries. We hypothesized that gene expression in human blood vessels is subject to circadian regulation. To investigate whether circadian genes oscillate in human blood vessels over time, we extracted RNA from saphenous veins and analyzed the ex‐vivo mRNA expression of clock genes for 24 hours at 3 hour interval. Surprisingly we found that the expression of core circadian genes failed to oscillate in saphenous veins. Studies in mice have shown that oscillation in gene expression in arteries is more robust than veins. Future studies are planned with human mammary arteries to confirm this in humans. To investigate the molecular regulation of circadian rhythms in vitro, we have generated circadian reporter cells using a bmal1‐luc lentivirus. In U20S cells we have recorded robust changes in luminescence over a 24 hour cycle. We plan to employ this lentivirus to explore the differences in circadian rhythmicity in human endothelial cells from different vascular beds.
Exogenous Tetrahydrobiopterin Restores Cutaneous Vasodilation in Hypercholesterolemic Humans by Stabilizing eNOS
Authors: Kutz Jessica Leigh , Smith Caroline J. , Holowatz Lacy A. , Kenney W. Larry
Published: June 2021

Local perfusion of exogenous BH4 corrects the deficit in heat‐induced vasodilation (VD) in hypercholesterolemic human skin through one of two plausible mechanisms: by stabilizing eNOS or through generalized antioxidant effects. We used the stereoisomer S‐BH4, which has the same antioxidant properties but does not function as a NOS cofactor, to elucidate the mechanism by which BH4 restores cutaneous VD in this population. Microdialysis fibers were placed in the skin of 17 normocholesterolemic (NC) and 15 hypercholesterolemic (LDL 91± 3 vs. 189±5 mg/dl) men and women to infuse Ringers (control site) and R‐BH4. In 6 NC and 6 HC subjects, S‐BH4 was dialyzed at a third site. Skin blood flow was measured during local heating. After cutaneous vascular conductance (CVC) plateaued, the NO‐dependent proportion of the total VD was quantified by perfusing L‐NAME (ΔNO). Data were normalized as %CVCmax. Fully‐expressed VD (NC: 98±2 vs. HC: 72±5%CVCmax) and the NO‐dependent portion (NC: 63±4 vs. HC: 47±4%CVCmax) were reduced in HC (both p<0.001). R‐BH4 increased full (94±4%CVCmax; p<0.001) and NO‐dependent VD (59±6%CVCmax; p<0.001) in HC but not in NC. S‐BH4 increased full (88±6%CVCmax, p=0.04) but not NO‐dependent vasodilation (37±9%CVCmax; p=0.3) in HC. Exogenous BH4 restores NO‐dependent VD in hypercholesterolemic human skin through NOS coupling mechanisms, not through generalized antioxidant properties.
Heat acclimatization in semi professional soccer players
Authors: Racinais Sebastien , Mohr Magni , Buchheit Martin , Voss Sven , Nybo Lars
Published: June 2021

This study aimed to determine the relationship between the individual heat acclimatization and field performance in 19 semi professional soccer players. A soccer match was played in a hot environment after a week of training in hot ambient conditions (environmental temperature: 38–43ºC). At the beginning and end of the week, all players performed a heat‐response test (30 min walk at 5 km.h‐1 + 30 min seated; 44ºC, 44% RH). Results from the heat response test showed an improvement (P < 0.05) in the physiological markers of heat acclimatization (sweat rate +34%, sweat sodium concentration −18%, plasma volume +4.3%). Despite these adaptations, the mean amount of activity during the match in hot was lower than during a control game played in thermo‐neutral conditions the week before (−6.0%, P < 0.05). The decrement in total distance during the game in the heat was correlated to the plasma volume expansion (r = 0.75, P < 0.05) and the players with the highest plasma volume expansion were able to maintain the same level of activity in the heat as in thermo‐neutral environment. These data suggest the existence of ‘responders’ and ‘non/slow ‐ responders’ to heat acclimatization that could be identified by a heat‐response test.
Nesfatin‐1 acts through a central POMC‐CRH‐OT circuit to increase mean arterial pressure
Authors: Yosten Gina L. C. , Samson Willis K.
Published: June 2021

Nesfatin‐1, a recently discovered brain/gut peptide, was originally described to potently inhibit food intake through interaction with the central melanocortin system (CMS), the central oxytocin system (COTS), and neurons producing corticotrophin releasing hormone (CRH). We previously have shown that central injection of nesfatin‐1 led to an increase in mean arterial pressure (MAP) that was also dependent upon the presence of functional central melanocortin and oxytocin receptors, and hypothesized that the cardiovascular effect of nesfatin‐1 would be dependent upon CRH neurons as well. We therefore pretreated conscious male rats with the CRH receptor antagonist, astressin‐2B, before treatment with nesfatin‐1, and found that blockade of CRH receptors abolished the ability of nesfatin‐1 to increase MAP. Several lines of evidence suggest that COT and CRH systems can act as downstream effectors of the CMS; however, little evidence exists as to the order of activation of oxytocin and CRH systems in this circuit. Pretreatment with the OT receptor antagonist, OVT, prior to central administration of a pressor dose of CRH, abrogated the CRH‐induced increase in MAP. Thus we have gained insight not only into the mechanism of nesfatin's central hypertensive actions, but also the hierarchy of neural networks within the hypothalamus projecting to brainstem cardiovascular centers. Supported by NIH #HL66023.
Influence of body mass index on neural cardiovascular reactivity to mental stress in humans
Authors: Durocher John J , Carter Jason R
Published: June 2021

A limited number of studies suggest an association between body mass index (BMI) and neural cardiovascular reactivity to mental stress in humans, but this relationship has not been comprehensively examined. We tested the hypothesis that overweight subjects (BMI ≥ 25 kg/m2) would demonstrate augmented neural and cardiovascular responses to mental stress when compared to normal weight individuals. Retrospective analysis of studies with recordings of heart rate (HR), mean arterial pressure (MAP), and muscle sympathetic nerve activity (MSNA) during 5 min of mental stress (via mental arithmetic) yielded 60 normal weight (age 23±1 yr; BMI 22±0 kg/m2) and 61 overweight (age 24±1 yr; BMI 28±0 kg/m2) subjects. Resting HR and MSNA were similar between normal and overweight subjects, while resting MAP was higher in overweight subjects (77±1 vs. 84±1 mmHg; P<0.05). MAP (Δ11±1 vs. Δ13±1 mmHg), HR (Δ18±1 vs. Δ18±1 beats/min), and MSNA (Δ4±1 vs. Δ5±1 bursts/min) responses to mental stress were similar between normal and overweight participants, respectively. In conclusion, our findings indicate that being classified as ‘overweight’ according to BMI standards does not increase neural or cardiovascular reactivity to mental stress. However, it remains unclear if more specific classifications of obesity (i.e., fat composition and distribution) influence neural cardiovascular reactivity to mental stress. Supported by NIH.
Role of cardiac output vs. peripheral vasoconstriction in mediating the muscle metaboreflex pressor response during dynamic exercise and post‐exercise muscle ischemia
Authors: Spranger Marty D. , Sala-Mercado Javier A. , Coutsos Matthew , Kaur Jasdeep , Stayer Douglas , O'Leary Donal S.
Published: June 2021

Muscle metaboreflex activation (MMA) during sub‐maximal dynamic exercise in normal individuals increases mean arterial pressure (MAP) via increases in cardiac output (CO) with little peripheral vasoconstriction. The rise in CO occurs primarily via increases in heart rate (HR) with maintained or slightly increased stroke volume. When the reflex is sustained during recovery (postexercise muscle ischemia ‐ PEMI), HR declines yet MAP remains elevated. The role of CO in mediating the pressor response during PEMI is controversial. In five chronically instrumented canines, steady state values with MMA during mild exercise (3.2 kph) were observed by reducing hindlimb blood flow by ~60% for 3–5 minutes. MMA was followed by 60 seconds of PEMI. Control experiments consisted of normal exercise and recovery. MMA during exercise increased MAP, HR and CO by 61.4 ± 4.0 mmHg, 47.1 ± 7.8 bpm, and 2.71 ± 0.43 l/min, respectively. During sustained MMA via PEMI, MAP remained elevated and CO remained well above the normal recovery levels. Neither MMA during dynamic exercise nor during PEMI significantly affected peripheral vascular conductance. We conclude that the sustained increase in MAP during PEMI is driven by a sustained increase in CO not peripheral vasoconstriction. HL‐55743 and HL‐095819
The effect of chronic treatment with diazepam on stress and hypertension in Schlager BPH/2J hypertensive mice
Authors: Davern Pamela , Chowdhury Sara , Jackson Kristy , Nguyen-Huu Thu-Phuc , Head Geoffrey
Published: June 2021

Schlager hypertensive mice (BPH/2J) are a neurogenic model of hypertension that show a greater BP increase with the onset of the dark when they become active. This induces greater neuronal activation (as detected by Fos) in the medial amygdala (MeAm), central amygdala, paraventricular hypothalamus and dorsomedial hypothalamus of BPH/2J compared with normotensive BPN/3J mice. Aversive stress (dirty‐cage switch) increased MAP in BPH/2J more than in BPN/3J and also produced greater Fos expression in the hypothalamus and amygdala. MeAm neurons are tonically inhibited by GABAergic interneurons and lesions of the MeAm reduce hypertension in BPH/2J mice. Diazepam acts via GABAA receptors to induce anxiolytic effects via fibres innervating GABAergic neurons. Together this suggests that BPH/2J hypertension could be due to a lack of GABA activation of the MeAm. Therefore, we investigated whether stress activated neurons in the MeAm receive less GABA inhibition and if this can be restored by chronic GABAA activation with treatment of diazepam. We used male BPN/3J and BPH/2J mice that were implanted with telemetry probes and recordings of cardiovascular and activity parameters were made before and after diazepam (2.5mg/kg/day) or vehicle administration for 7 days. The results show that chronic diazepam treatment in BPN/3J mice (n=7) decreased MAP (−4.8 mmHg; P<0.01), while BPH/2J mice (n=7) had no significant change in MAP (+2.1mmHg). Restraint stress in diazepam treated mice reduced BP and was associated with fewer activated neurons (Fos) in the MeAm in BPN/3J mice (18±1.2) compared with BPH/2J mice (25±1.2; P<0.01). In the MeAm of BPH/2J mice, 68% of activated neurons (Fos) also expressed GABAA receptors, with no colocalisation observed in BPN/3J mice. Together our data suggests a fundamental irregularity of GABA inhibitory regulation in the MeAm of BPH/2J hypertensive mice.
The 2‐nitrate‐1,3‐dibuthoxypropan, a nitric oxide donor, alters autonomic function in spontaneously hypertensive rats
Authors: Silva Maria Socorro França , Monteiro Matheus M. O. , Medeiros Isac A. , Braga Valdir A.
Published: June 2021

The 2‐nitrate‐1,3‐dibuthoxypropan (NDBP), an organic nitrate synthesized from glycerin, increases NO levels in rabbit aortic smooth muscle cells and causes a biphasic response: hypotension and bradycardia followed by hypertension and tachycardia in Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. However, its effect on autonomic control has not been investigated. This study evaluated the action of NDBP on autonomic function in SHR and WKY rats. Atropine (2 mg/kg) blunted the bradycardia induced by NDBP (15 mg/kg) in WKY and SHR (−265±25 vs −48±7 bpm, n=6 and −255±9 vs −25±10 bpm, n=6; p<0.05). Furthermore, the pressor response to the compound was potentiated (35±7 vs 65±4 mmHg in WKY; 33±10 vs 82±6 mmHg in SHR; p<0.05). Vagotomy also reduced the bradycardia in both WKY and SHR (−136±8 vs −17±2, n=4 and −171±36 vs −8±2, n=6, p<0.05). Moreover, hexamethonium (30 mg/kg) reduced both bradycardia (−261±10 vs 1±3 in WKY; −282±21 vs −26±25 in SHR, n=4; p<0.05) and pressor response (41±4 vs 0±1‐in WKY; 44±9 vs −12±7 in SHR, n=4; p<0.05) elicited by NDBP. In conclusion, NDBP, in addition to its effects as NO donor, induces cardiovascular effects by altering autonomic function.
Physical (In)activity dependent changes in the morphology of RVLM neurons
Authors: Mischel Nicholas A , Llewellyn-Smith Ida J , Mueller Patrick J
Published: June 2021

The rostral ventrolateral medulla (RVLM) contains neurons critical for cardiovascular regulation. Functional plasticity in RVLM neurons may contribute to increased sympathoexcitation in disease states. Here, we aimed to define morphological differences in RVLM cardiovascular neurons in sedentary vs physically active rats, hypothesizing that increased sympathoexcitation in sedentary rats would correlate with more dendritic branching. Physically active rats were provided with in‐cage running wheels for 14 weeks and ran 230 ± 81 km (n=4). We used immunohistochemistry for tyrosine hydroxylase (TH) in 150 μm brainstem sections and tracing software to reconstruct TH‐immunoreactive neurons at the caudal pole of the facial nucleus. Comparison of dendrites in 3 sedentary vs 4 active rats showed an average length of 1111±54 μm vs. 686±86 μm (p<0.05) and 9.7±0.7 branch nodes vs. 7.0±1.1 (p=0.058). A Sholl analysis showed 82±4.2 vs. 52.8±6.2 total intersections (p<0.05). These data suggest that greater dendritic branching in RVLM cardiovascular neurons contributes to increased sympathoexcitation and incidence of cardiovascular disease in sedentary individuals. (F30‐HL105003; NHMRC 480414; R01‐HL096787).
Autoradiography of non‐AT‐1, non‐AT‐2 125‐I‐SI angiotensin II binding in neurolysin knock‐out and wild‐type mouse brains
Authors: Speth Robert C , Santos Kira L , Swindle Jamala D. , Reiley Luz Gonzalez , Linares Andrea , Schadock Ines , Bader Michael , Karamyan Vardan T
Published: June 2021

Recently a novel, non‐AT1, non‐AT2 binding site for angiotensin II (Ang II) was identified as the metalloendopeptidase neurolysin. To assess the localization of this binding site in brain, 3 mice deficient in neurolysin and 3 wild‐type mouse brains were evaluated for radioligand binding with 125I‐Sar1, Ile8 Ang II (250 pM) in the presence of AT1 and AT2 receptor‐saturating concentrations of losartan and PD123319, and 150 μM p‐chloromercuribenzoate using in vitro autoradiography. Specific (10 μM Ang II displaceable) 125I‐Sar1, Ile8 Ang II binding in wild‐type mouse brains was abundant, with highest levels in the molecular layer of the cerebellum, cerebral cortex, hippocampus, amygdala, caudate‐putamen, hypothalamus, lateral septum, and external plexiform layer of the olfactory bulb. Specific 125I‐Sar1, Ile8 Ang II binding in the neurolysin‐deficient mouse brains was profoundly reduced, however, the extent of reduction was region‐specific. Large decreases were seen in the cerebral cortex, substantia nigra, hippocampus, paraventricular thalamus, lateral septum, nucleus accumbens. Cerebellar cortex and hypothalamus showed moderate reduction in binding. These results confirm neurolysin as the novel non‐AT1, non‐AT2 receptor binding protein, but may reveal additional non‐AT1 non‐AT2 binding sites in the mouse brain. Supported by NHLBI HL‐096357
Developmental Time‐course of Vascular RNA Expression and Protein Levels for ACE, eNOS and iNOS in Young Syrian Cardiomyopathic Hamsters
Authors: Cruz Nildris , Quidgley Jose , Garcia Juan M. , Torres Giselle M. , Escobales Nelson , Miranda Jorge D. , Altieri Pablo I. , Crespo Maria J.
Published: June 2021

Endothelial dysfunction, increased oxidative stress, and alterations in the renin‐angiotensin system (RAS) are present in 2‐month‐old Syrian cardiomyopathic hamsters (SCH). To evaluate the developmental time‐course of these alterations, we assessed the protein levels and RNA expression of ACE, eNOS and iNOS in the vasculature of SCH from 1 to 4 months of age. Age‐matched golden hamsters were used as controls (CT). Total RNA and protein were extracted from aortic tissue. RNA expression was quantified with Real‐Time RT‐PCR, and protein levels were determined with Western blot. We found that ACE RNA was upregulated in 2‐month‐old SCH (from 38.85% to 141.18 %, P<0.05), when compared with 1‐month‐old SCH, and reached a plateau at 4 months. ACE protein levels were higher at 2‐months (1.54 arbritrary units; AU) than at 1 month (0.98 AU) and 4 months (.79 AU). In addition, eNOS RNA increased significantly in SCH from 1 to 4 months (from 49 % to 273%; P<0.05), but eNOS protein levels decreased after 1 month (1.06 AU), reaching its minimum at 2 months (0.34 AU, P<0.05). Vascular iNOS RNA and protein levels, by contrast, remained unchanged in SCH from 1 to 4 months. These results suggest that the increased ACE RNA and protein levels, together with the decreased eNOS protein content observed at 2 months, may be involved in the etiology of endothelial dysfunction that is present in 2‐month‐old SCH. Supported by MBRS‐RISE R25‐GM061838.
Effect of brain specific deletion of PPARγ on puberty onset, hypothalamic gene expression and diet induced obesity (DIO) in C57BL/6 mice
Authors: Fernandez Marina Olga , Sharma Shweta , Hwang Vicky , Olefsky Jerrold M. , Webster Nicholas J.
Published: June 2021

Proliferator‐activated receptor gamma (PPARγ) ligands have been used for the treatment of insulin resistance in type 2 diabetes and polycystic ovary syndrome. We created a neuronal‐specific deletion of PPARγ (BKO) by crossing a floxed PPARγ mouse to a Synapsin‐cre mouse and assessed the effect of the gene deletion on reproduction. Male and female BKO mice exhibited reduced litter sizes; females also showed elevated pituitary LHβ mRNA and serum LH levels and hemorrhagic corpora lutea with reduction of primary and secondary follicles in the ovary. Male BKO mice also showed increased tubular degeneration in the testis. Female BKO mice did not exhibit alterations in the onset of puberty, nor did we observe alterations in hypothalamic GnRH in proestrus or estrus, or Kiss1 and TRH mRNA in estrus. We have previously published that BKO male mice are resistant to diet‐induced obesity (DIO) and leptin‐sensitive. Female BKO and control littermates do not show differences in their fasting basal glucose levels or glucose tolerance tests when fed normal chow, similar to the male BKO mice. The effects of DIO on reproduction are being assessed by putting BKO and control mice on a 60% high‐fat diet or a 10% fat control diet, and we will present data on glucose and leptin sensitivity, as well as basal and GnRH‐stimulated gonadotropin levels and estrous cyclicity to further understand the role of brain PPARγ in modulating reproduction. Supported by NIH grant U54HD12303.
Differential Responses of Genes for Neurosecretory Granules in the Rat Adrenal Medulla to Acute and Repeated Stress
Authors: Sabban Esther Louise , Tillinger Andrej , Serova Lidia I , Nostramo Regina
Published: June 2021

With acute stress, release of adrenomedullary catecholamines is important for handling the emergency situation. However, when chronic or repeated, stress leads to development or worsening of wide range of diseases. To clarify the mechanism, we studied changes in components of neurosecretory granules in adrenal medulla of male Sprague Dawley rats exposed to immobilization stress for 2 h once (1xIMO) or for 6 days (6xIMO). While vesicular monoamine transporter 1 (VMAT1) was expressed in all chromaffin cells, VMAT2, which has greater affinity for catecholamines, and higher turnover number than VMAT1, was expressed only in the NE‐cells under basal conditions. 1xIMO elevated VMAT2 mRNA, and after 6xIMO, this was reflected in nearly 3‐fold rise in VMAT2 protein, and expression in many Epi‐synthesizing cells. With 6xIMO, not only was VMAT2 mRNA still elevated, but chromogranin A (CgA) and chromogranin B (CgB) mRNAs were also increased 2–3 fold. The elevation of CgA, CgB and VMAT2 are each expected to increase the quantal size. This can facilitate more efficient use of the well characterized heightened catecholamine levels. To investigate a possible role of the HPA axis in induction of VMAT2, PC12 cells were treated with dexamethasone, which elevated VMAT2 mRNA expression. In contrast, the rise in Egr1 and P‐CREB may mediate the induction of CgA and CgB with repeated IMO stress.

American Heart Association 10GRNT4420001
The full‐length estrogen receptor ERα66 mediates ERK activation and downregulation of ERα36 and ERβ expression in kidneys of female diabetic mice
Authors: Irsik Debra Lima , Fallet Rachel W. , Carmines Pamela K. , Lane Pascale H.
Published: June 2021

The kidney expresses multiple estrogen receptors, including the full‐length ERα66, its ERα36 splice variant, and ERβ. We previously reported that female ERα66KO mice (which lack a functional ERα66, but still express ERα36) were protected from diabetes‐induced glomerular enlargement, an early marker of renal complications. We hypothesized that diabetes triggers ER‐dependent pro‐hypertrophic signaling events, in concert with altered ER protein levels. Kidneys were harvested 2 wks after inducing type 1 diabetes (streptozotocin injection) in female wild‐type (WT) and ERα66KO mice, with insulin provided to temper the degree of hyperglycemia; sham mice received vehicle treatments. Western blot analysis revealed that phosphorylated‐to‐total ERK1/2 was increased in kidneys from diabetic WT mice, averaging 170±34 % of sham WT (P<0.05); however, this parameter was unaltered by diabetes in ERα66KO mice (97±13 % of sham ERα66KO). Renal ERα36 and ERβ protein levels were reduced in diabetic WT mice, averaging 42±7 and 43±5 % of sham WT values, respectively (both P< 0.05). In contrast, diabetes did not significantly decrease renal ERα36 and ERβ levels in ERα66KO mice. In summary, the renal ERα66‐dependent events triggered by diabetes include downregulation of both ERα36 and ERβ, as well as ERK activation. These processes may contribute to the glomerular enlargement that arises early in diabetes.
Sildenafil Administration Attenuates Placental Ischemia and sFlt‐1 Induced Hypertension in Pregnant Rats
Authors: George Eric M , Cockrell Kathy , Arany Marietta , Granger Joey P
Published: June 2021

Preeclampsia is a complication of pregnancy which is marked by hypertension, proteinuria, and maternal endothelial dysfunction. A central factor in the etiology of the disease is the development of placental hypoxia/ischemia, which results in release of pathogenic soluble factors. There is currently no effective treatment for preeclampsia, but one proposed therapeutic agent is the phosphodiesterase‐5 (PDE‐5) inhibitor sildenafil, as PDE‐5 is enriched in the uterus, and its antagonism could improve uteroplacental perfusion. Here we report in two rodent models that administration of oral sildenafil is effective in attenuating hypertension during gestation. In the RUPP placental ischemia model, arterial pressure is increased significantly when compared to control animals (132±3 vs 100±2 mmHg, p<0.05). Administration of sildenafil (45mg/kg/day) in drinking water had no effect on blood pressure in control rats, but decreased pressure in RUPP rats (115±1mmHg, p<0.05). Similarly, in the sFlt‐1 infusion model of hypertension, pressure is increased significantly over control rats (116±2 vs 99±2mmHg, p<0.05). In these rats sildenafil administration essentially normalized blood pressure (103±2, p<0.05 vs sFlt‐1 infused alone). These data suggest that administration of sildenafil might prove an effective therapeutic in the management of hypertension during preeclampsia.
Hyperinsulinemia increases blood pressure and pup weight in pregnant rats
Authors: Palei Ana Taveiros , George Eric , Cockrell Kathy , Arany Marietta , Granger Joey
Published: June 2021

Hyperinsulinemia and insulin resistance have been associated with gestational hypertensive disorders. The aims of this study were to investigate whether chronic hyperinsulinemia during euglycemia causes changes in cardiovascular, metabolic and reproductive systems of pregnant rats. On gestational day (GD) 14, Sprague Dawley rats were assigned to normal pregnant (NP) group or to NP+Ins group, in which miniosmotic pump with insulin (1.5 mU/kg/min) was placed subcutaneously and 20% glucose was added in drinking water. On GD 19, MAP was recorded, rats were sacrificed, and blood, placentas and pups were collected. Body weight (BW) and caloric intake (CI) were measured on GD 16–18. Serum insulin concentration was elevated in NP+Ins compared with NP (15.61 ± 1.39 vs 10.56 ± 0.68 μU/mL, P<0.05). There were no differences in blood glucose levels between groups. MAP was higher in NP+Ins compared with NP (114 ± 2 vs 104 ± 4 mmHg, P<0.05). There were no differences in BW between groups. CI was lower in NP+Ins compared with NP (55.24 ± 1.32 vs 69.98 ± 1.35 kcal/day, P<0.05). Although number of viable pups per rat and placenta weight were similar between groups, pups from NP+Ins were heavier than from NP (2.41 ± 0.02 vs 2.32 ± 0.03g, P<0.05). Thus insulin appears to raise blood pressure, suppress appetite and increase pup weight in pregnancy, representing a possible mechanism to induce hypertension in gestational hypertensive disorders. Funded by NIH grants HL51971 and 1T32HL105324.
Physiological genomics: Renal gene interaction in diabetic nephropathy
Authors: Kelly Katherine J , Dominguez Jesus
Published: June 2021

Diabetic nephropathy (DN), the most common cause of chronic kidney failure (CKD), is accelerated by ischemic acute kidney injury (AKI) to end‐stage renal disease (ESRD). We hypothesize that renal transcriptional analysis by RNAseq can reveal the interacting genes of DN. We studied 3 groups of ZSF1 rats from 8 to 25 weeks of age: lean sham operated controls (LS, n = 6, 4 transcriptomes, “tomes”), sham operated obese/diabetic (DS, n = 7, 4 tomes), and obese/diabetic subjected to renal ischemia for 25 min at 10 weeks of age (DI, n = 11, 4 tomes). DS and DI had progressive CKD, but CKD progression was significantly worse in DI. The 436 × 50 bp reads (per each of 12 kidneys) were aligned to a database and analyzed. There were 16,536 sequenced genes per kidney, some verified by RT‐PCR. There were 1442 upregulated genes in DI compared to LS and 184 up‐regulated genes in DS compared to LS. There were 1239 down‐regulated genes in DI compared to LS. In DS, 396 genes were down‐regulated compared to LS. Gene pathways strongly and significantly assigned up‐regulated genes to inflammatory pathways in DI and DS. We conclude that AKI‐induced rapid CKD progression in DI is accompanied by a larger number of up‐regulated and down‐regulated genes, even 15 weeks of post‐ischemia AKI. We propose that deep sequencing yields critical genetic information in rapidly progressive DN, and may serve as a template for intervention in progressive CKD.
The different mode of organic anion transport in NPT1/SLC17A1 and NPT4/SLC17A3
Authors: Jutabha Promsuk , Endou Hitoshi , Anzai Naohiko
Published: June 2021

Voltage‐driven organic anion transporter has been hypothesized at the apical side of renal proximal tubule based on the classical membrane vesicle study. We first clarified its molecular identity (OATv1) in pig kidney, and searched its human ortholog. Human NPT1 (SLC17A1) has the highest identity (63%) to amino acid sequence of pig OATv1, but does not mediate voltage‐driven transport. Recently, we identified NPT4 (SLC17A3) as a human voltage‐driven organic anion transporter in kidney. To further clarify the difference of NPT1 and NPT4, we injected their cRNAs into Xenopus oocytes and compared transport properties. NPT1 showed broader substrate recognition for organic anions than NPT4. Estrone sulfate is a new substrate of NPT1 similar to NPT4. The major transport modes are different: Cl− exchange for NPT1 and voltage‐driven for NPT4. Both modes are observed in pig OATv1‐mediated transport. Deletion of PDZ‐motif at C‐terminus of NPT1, not in NPT4, abolished its transport function, suggesting that PDZ‐domain containing protein may regulate NPT1 function. The co‐localization of NPT1 and NPT4 at apical membrane of renal proximal tubules may be useful for the efficient organic anion secretion in kidney.