bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of cysteine residues for the function of the human sodium‐dependent multivitamin transporter (hSMVT)
Authors: Ghosal Abhisek , Said Hamid M
Published: June 2021

The hSMVT plays an important role in biotin uptake. While significant knowledge has been gained for regulation and cell biology of SMVT, there is little known about its structure‐functional activity relationship. Using site‐directed mutagenesis we examined the role of each of the ten conserved cysteine residues in the function of hSMVT. The results showed that mutation at Cys294 only (but not at the other conserved cysteine residues) significantly impairs biotin transport and the effect is not related to polar status of substituting amino acids. Kinetic studies showed the inhibition to be mediated via a significant reduction in the Vmax, but not the apparent Km, of the biotin uptake process, suggesting a decrease in the number (and/or activity) of hSMVT but not its affinity. Biotinylation assay confirmed the latter observation by showing a marked reduction in the level of expression of the mutated hSMVT protein at the cell membrane. Mutation of non conserved Cys at position 628 also showed inhibition in biotin uptake suggesting possible interaction with Cys294. These results show an important role for Cys294 in the function and cell biology of hSMVT.

(Supported by grants from the DVA and the NIH)
Regulation of ENaC by Cholesterol: Role of Microvilli and PIP2
Authors: Ma He-Ping
Published: June 2021

We previously showed that cholesterol (Cho) stimulates the renal epithelial sodium channel (ENaC). However, the underlying mechanism remains unclear. Using the cell‐attached configuration, we found that depletion of Cho in the outer leaflet of A6 cell apical membrane with methyl‐β‐cyclodextrin (MβCD) reduced the number of ENaC, which is consistent with the data from immunohistochemical staining and biotinylation of ENaC. Atomic force microscopy scanning of A6 cell apical membrane showed that MβCD induced destruction of microvilli. These data indicate that depletion of Cho reduces the number of ENaC probably by causing destruction of microvilli. Further, confocal microscopy data showed that ENaC is co‐localized with both Cho and PI(4,5)P2 in the microvilli and that depletion of Cho reduced PI(4,5)P2. In inside‐out patches, MβCD significantly reduced ENaC open probability (PO); the effect was reversed by Cho. However, MβCD and Cho failed to regulate ENaC when PI(4,5)P2 is sequestered with its antibody. Since ENaC is mainly located in microvilli and stimulated by PI(4,5)P2, these data suggest that depletion of Cho reduces ENaC density via destruction of microvilli and decreases ENaC PO via reduction of PI(4,5)P2.
Renal hemodynamics in the anesthetized giraffe
Authors: Bie Peter , Grøndahl Carsten , Bertelsen Mads Frost , Hørlyck Arne , Hasenkam J. Michael , Wang Tobias , Brøndum Emil Toft , Candy Geoffrey , Kristensen Bodil Aarup
Published: June 2021

In the giraffe, the brain is located some two meters above the heart most of the time and the central mean arterial blood pressure is twice that of mammals of similar mass. Consequently, the heart perfuses the kidney at a mean pressure of some 200 mmHg. The renal capsule is thick and non‐translucent.

Nine giraffes (mean body mass 493 kg) were anaesthetized and instrumented. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were determined by conventional clearance techniques using continuous infusions of inulin and p‐aminohippurate. Renal interstitial hydrostatic pressure (RIHP) was measured by a 22G needle inserted in the renal parenchyma. The intrarenal artery resistance index (AI) was determined by Doppler ultrasound. Hormone immunoactivities in plasma were measured by conventional radioimmunoassays.

GFR was 342 ± 99 ml/min and ERPF 1252 ± 305 ml/min. AI was median 0.27 (max. 0.49, min. 0) and RIHP 142 ± 6 mmHg. Plasma renin concentration was 17 ± 4 mIU/l, peptide levels were 6.3 ± 0.9 pg/ml for AngII, 135 ± 23 pg/ml for atrial natriuretic peptide, and highly variable for vasopressin.

The data show low values of GFR, ERPF and resistance index, yet the RIHP is >20 fold higher than in other mammals. The results suggest that a strong renal capsule allows for a high RIHP reducing the net arterial driving pressure and effectively protecting the kidney against organ damage.
Natriuretic response to renal medullary endothelin B receptor activation is impaired in Dahl‐salt sensitive rats on a high‐caloric diet
Authors: Kittikulsuth Wararat , Zhang Yawen , Sunjic Igor , Pollock David M.
Published: June 2021

Renal medullary endothelin B receptors (ETB) mediate sodium excretion and blood pressure (BP) control. Several animal models of hypertension including the Dahl salt sensitive (DS) rat on a high salt diet have impaired renal medullary endothelin system. Our lab has recently reported that the DS rat becomes hypertensive on a normal salt, high‐caloric diet. Thus we hypothesized that renal medullary ETB function is dysfunctional in DS rats fed a high‐caloric diet. The diuretic and natriuretic response to intramedullary infusion of ETB agonist sarafotoxin 6c (S6c) was determined in DS rats fed either a normal (14% fat; 3.93 kcal/g) or high‐caloric diet (59% fat; 5.45 kcal/g) for 4 weeks. We found that 4‐week high‐caloric diet elevated systolic BP in DS rats (126±2 vs. 143±3 mmHg, p<0.05). In DS rats fed a regular diet, intramedullary infusion of S6c (0.45 μg/kg/hr) increased urine flow (3.6±0.5 μl/min during baseline vs. 7.0±0.5 μl/min after S6c; p<0.05) and sodium excretion (0.42±0.09 vs. 0.97±0.08 μmol/min; p<0.05). However, DS rats fed a high‐caloric diet did not display an increase in urine flow (2.8±0.3 vs. 4.3±0.5 μl/min) or sodium excretion (0.35±0.07 vs. 0.48±0.12 μmol/min) upon S6c infusion. These data demonstrate that renal medullary ETB function is impaired in DS rats fed a high‐caloric diet, which may contribute to the elevation of blood pressure during high‐caloric feeding in this model.
Kidney function and sulphate transport in Pacific Hagfish (Eptatretus Stoutii)
Authors: Goss Greg G , Schultz Aaron , Goss Brendan L , Clifford Alexander M
Published: June 2021

The hagfish diverged from vertebrate lineage ~550–600 mya and therefore represent a unique model to study early ionoregulatory strategies. Whilst hagfish are osmoconformers for Na+ and Cl−, these animals have plasma concentrations of the divalent (Ca2+, Mg2+ and S042‐) ions at 1/3 to 1/2 seawater. A difficulty in molecular research involving hagfish is the lack of genomic information. Using a recently obtained hagfish Illumina transcriptome, we have identified a putative sulfate transporter (SLC26a1). We investigated if hagfish possess active S042‐ handling mechanisms by injecting a chronic (3 day) S042‐ load to upregulate potential S042‐ handling and transporting abilities. Using 3H‐ inulin and 35S (as S042‐) injections into the blood, we monitored both GFR and net S042‐ excretion. We also investigated the changes in expression of hfSLC26a1. We determined that Pacific hagfish have a relatively high GFR (~ 0.46 mls/kg/h) despite being osmoconformers and are able to increase sulfate excretion/handling following chronic S042‐loading from 0.06 umol/kg/h to 0.28 umol/kg/h.
Fluctuations in oxygen influences facultative endothermy in bumblebees
Authors: Dzialowski Edward M , Tattersall Glenn , Nicol Stewart , Frappell Peter
Published: June 2021

Bumblebees are facultative endotherms, having the ability to elevate thorax temperature above ambient temperature by elevating metabolism. Here we investigated the influence of hypoxia on metabolic demands and thermoregulatory capabilities of the bumblebee Bombus terrestris. We measured thorax temperature, oxygen consumption, carbon dioxide production, and ventilation rates of bees exposed to oxygen levels of 21, 15, 10, and 5 kPa at 26°C. Under normoxia, bumblebees maintained an elevated thorax temperature of 35.5 °C. There was no significant change in thorax temperature at 15 kPa oxygen (33.4°C). Mean thorax temperature decreased significantly at 10 kPa (31.6 °C) and 5 kPa (27.3 °C). Bees were able to maintain an elevated metabolic rate at 15 kPa and 10 kPa. In normoxia, thermoregulating bees exhibited periods of rapid abdominal pumping (327 s−1) interspaced by periods of no abdominal pumping. At 10 kPa oxygen, abdominal pumping rate deceased (255 s−1) but became continuous. Upon exposure to 5 kPa, metabolic rate and abdominal pumping rate (152 s−1) dropped rapidly although the animals continued abdominal pumping at the reduced rate throughout the exposure period. Bumblebees are able to meet the energetic demands of endothermy at 15 kPa oxygen, but begin to become compromised at levels of 10 kPa oxygen.
Can edge triggering response offer precious information of the visual motor response?
Authors: Kim Doh-Yeon , Choi Han-Kyun , Jung Chang-Sub
Published: June 2021

It has been known that olfactory stimuli increase the visual sensitivity in the fly and the fish. In flies, odorant caused flies both to increase aerodynamic power output and to steer straighter. In fish, visual motor response was increased upon presentation of amino acids as olfactory stimuli. In this study, we analyzed the increasing mechanism of visual motor response of the goldfish with measuring the optomotor response (OMR), edge triggering response (ETR) and electroretinogram (ERG). Before and after olfactory stimulation, we measured the OMR and the ETR. The goldfish's food solution and four amino acids (L‐alanine, L‐methionine, L‐arginine and L‐aspartic acid) were used for olfactory stimulation. The effect of amino acids on ERG at photopic level was determined before and after amino acid administration into one side of the fish's nostril. Olfactory stimulation by food solution increased the OMR up to about 20%. Olfactory stimulation by amino acids almost increased the OMR. ETR, the specific behavior observed in goldfish when it triggers the edge of a moving pattern, decreased by odorant. These results indicated that olfactory stimuli minimize the loss of tracking the moving pattern and thereby increasing the trailing distance without increasing swimming velocity. There were no products of ERG increasing by the odorant at photopic light level. Here we report that olfactory stimulation increased the motivation of visual perception to rotational motion and thereby diminishing the loss of tracking. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011‐0004937). The authors wish to acknowledge the financial support of the Catholic Medical Center Research Foundation made in the program year of 2010.
Deficiency of p47phox subunit of NADPH oxidase protects skeletal muscle from depression of force stimulated by sphingomyelinase
Authors: Bost Elaina Reid , Ferreira Leonardo
Published: June 2021

Chronic diseases (e.g., heart failure) increase sphingomyelinase (SMase) activity in the serum and skeletal muscle. SMase forms ceramide, which activates reactive oxygen species production by mitochondria and NADPH oxidase. Reactive oxygen species contribute to depression of force stimulated by SMase. p47phox is a subunit required for activation of NOX2 isoform of NADPH oxidase. We hypothesized that mice lacking p47phox would be protected from depression of force when treated with SMase. We exposed diaphragm bundles from genetic controls (C57BL/6J; n = 16) and p47phox knockout mice (C57BL/6J; n = 11) to vehicle (glycerol) or SMase (0.5 U/ml). Maximal tetanic force decreased in genetic controls (in N/cm2: Vehicle 20 ± 0.6, SMase 16 ± 0.7, P < 0.05), but was unchanged in p47phox knockout (in N/cm2: Vehicle 20 ± 1.0, SMase 19 ± 1.1). Our results suggest that SMase‐induced depression of force requires signaling through the p47phox subunit of NADPH oxidase. It appears that SMase increases reactive oxygen species production by NADPH oxidase, which leads to depression of force.

Funding support: NIH 1K99HL098453‐01, 4R00HL098453‐02
Lipid stimulates myoglobin expression in skeletal muscle cells
Authors: Schlater Amber Elizabeth , De Miranda Michael Anthony , Corley Alyssa M , Kanatous Shane B
Published: June 2021

Myoglobin (Mb) is an oxygen‐binding hemoprotein generally localized in muscle tissue, where it functions as an oxygen store, nitric oxide (NO) scavenger, and reactive oxygen species (ROS) scavenger. Regulation of Mb is thought to occur through a calcium‐dependent signaling pathway involving calcineurin and nuclear factor of activated t‐cells (NFAT), with increases in Mb expression attributed to factors such as hypoxic exercise and NO. Here, we show a link between lipid supplementation and Mb expression in C2C12 cells. Specifically, C2C12 cells were cultured in normoxic (21% O2) or hypoxic (0.5% O2) conditions in either standard, high glucose media or a 5% lipid‐supplemented media. Mb assays and western blots revealed hypoxic cells differentiated in high fat media had significantly higher Mb content than high glucose hypoxic cells and both lines of normoxic cells. Citrate synthase activity (i.e. aerobic activity), however, significantly decreased in hypoxic, high fat cells. Further, normoxic cells were also compared to tissue from rats fed a high fat diet. Interestingly, both normoxic high fat conditions showed no change in Mb activity (per Mb assay) relative to the high glucose conditions, but did show an increase in total Mb protein (per western blot), suggesting an increase in non‐functional Mb. These results present a novel role for lipids in stimulating Mb, possibly to fill a niche beyond oxygen storage.
Fat accumulation, fibrosis, fiber‐type switching, and a reduction in specific force production following rotator cuff tear
Authors: Gumucio Jonathan , Davis Max , Stafford Patrick , Bradley Joshua , Schiffman Corey , Lynch Evan , Claflin Dennis , Bedi Asheesh , Mendias Christopher
Published: June 2021

Rotator cuff tears often cause pain and decreased mobility, and are associated with atrophy of muscle fibers, fibrosis and accumulation of fat in the muscle, collectively referred to as “fatty atrophy.” Previous studies have demonstrated that surgical repair of torn cuff muscles does not reduce fatty atrophy. As the molecular etiology of fatty atrophy remains unknown, gaining greater insight into the mechanisms that lead to the development of fatty atrophy will improve the treatment of rotator cuff tears. Using a rat model, we measured single fiber contractility, fiber type distribution, and expression of various mRNA and miRNA transcripts involved in lipid accumulation and matrix synthesis in torn and intact cuff muscles. We hypothesized that following chronic rotator cuff tear, there would be a decrease in specific force, accumulation of type IIb fibers, and an upregulation of adipogenic and fibrogenic genes. Thirty days after inducing the tear, cuff muscles had a substantial decrease in mass and force production, a dramatic accumulation of type IIb fibers, and a marked increase in fatty macrophages and caspase positive fibers. While there was an increase in several mRNAs and miRNAs associated with fibrosis, only modest changes in canonical adipogenesis transcripts were observed. Combined, the fatty atrophy phenotype in torn rotator cuff muscles may be a result of autophagy rather than bona fide adipogenesis.
Short‐term exercise training enhances nitric oxide‐mediated sympatholysis
Authors: Jendzjowsky Nicholas Gregory , DeLorey Darren Scott
Published: June 2021

We hypothesized that exercise training (ET) would enhance nitric oxide (NO)‐mediated inhibition of sympathetic vasoconstriction in contracting skeletal muscle. Sprague‐dawley rats (n=36) were randomly assigned to sedentary (S), mild‐ (M: 20m/min5% grade) or heavy‐intensity ET groups (H: 40m/min5% grade) and trained 5d/wk for 4 wks with the volume of ET matched. Rats were anesthetised and instrumented for stimulation of the lumbar sympathetic chain and the measurement of arterial blood pressure and femoral artery blood flow. The triceps surae muscles were stimulated to contract at 30 and 60% of maximal contractile force (MCF). The percent change in femoral vascular conductance (%FVC) in response to sympathetic chain stimulation (2 and 5 Hz) was determined at rest and during contractions before and after NO synthase (NOS) blockade. The magnitude of sympatholysis (%FVC rest – contraction) was greater in H compared to M and S at 30% MCF (2Hz‐ S: 9±5, M: 11±8, H: 18±7; 5Hz‐ S: 6±6, M: 12±9, H: 18±7; p<0.05) and was greater in H and M compared to S at 60% MCF (2Hz‐ S: 15±5; M: 25±3; H: 36±6; 5Hz‐ S: 22±6; M: 33±9; H: 39±9; p<0.05). NOS blockade did not alter the magnitude of sympatholysis in S during contraction at 30 or 60% MCF. In contrast, sympatholysis was diminished in H at 30% MCF and M and H at 60% MCF by NOS inhibition. These data demonstrate that short‐term ET augments sympatholysis through an NO‐mediated mechanism. NSERC Canada.
GABA‐A receptor inhibition reverses autoresuscitation defects in serotonin (5‐HT)‐deficient Pet‐1−/− mice
Authors: Cummings Kevin James , Li Aihua , Nattie Eugene
Published: June 2021

Autoresuscitation is compromised in serotonin (5‐HT)‐deficient Pet‐1−/− mice during a critical developmental period (P7‐10). We hypothesized that the autoresuscitation defects of Pet‐1−/− mice can be restored by antagonizing GABAA receptors. We gave an intracerebroventricular injection of either gabazine (2 uL of 20uM) or saline to Pet‐1−/− pups (n=5 and 7, respectively) and wild‐type littermates (WT; n=14 and 7) at P8. We tested autoresuscitation over 10 asphyxic episodes. We determined survival, delay to gasping, and time required for the recovery of heart rate and normal breathing. ~60% of WT animals survived all 10 episodes, while no Pet‐1−/− animals survived beyond 4 episodes (median=2). Lower survival in Pet‐1−/− was associated with a 7‐fold increase in the delay to the first gasp and a 3‐fold increase in the time required to restore heart rate upon return to room air. Gabazine completely reversed the autoresuscitation defects of Pet‐1−/− mice (P<0.001 for gasp delay and cardio‐respiratory recovery). The median number of asphyxic episodes survived by gabazine‐treated Pet‐1−/− mice was 4, with 2 of 5 surviving to the 8th episode. Based on these data we suggest that pharmacological inhibition of the GABA system could help reduce SIDS in infants that can be identified as being 5‐HT deficient.

This research was supported by a Parker B. Francis Fellowship (KJC) and NIH grant HL28066, (PI, E. E. Nattie)
Prenatal nicotine exposure increases frequency and duration of apneic events in neonatal rats
Authors: Kidder Ian Jeoffry , Mudery Jordan Andrew , Bailey E. Fiona
Published: June 2021

Prenatal nicotine exposure (PNE) has a profound effect on respiratory development and is linked to an increased risk for sudden infant death syndrome (SIDS) (DiFranza and Lew, 1995; Kinney et al., 1992). We tested the hypothesis that PNE decreases drive to muscles of the upper airway and increases the incidence and duration of apneic events. Sprague‐Dawley rats were exposed to nicotine (6mg/kg/day) in utero or remained unexposed. At ages 7–10 days, rat pups were anesthetized (inactin, 70mg/kg) and instrumented with fine wire electrodes in the genioglossus muscle of the upper airway and the intercostal muscles of the respiratory pump. Respiratory‐related chest expansion was monitored by a force transducer coupled to the chest wall. Rat pups were placed in a flow‐through chamber while activity was monitored under room air and normoxic hypercapnia (3%, 6%, and 9% FICO2). Our preliminary results reveal an increase in frequency and duration of apneic events in PNE animals compared to unexposed animals, while upper airway muscle activity remains unaffected. Such findings provide insight into the influence of nicotine on respiratory development in vivo and the potential complications leading to SIDS.
Peripheral Cardiac Sympathetic dysfunction in the prehypertensive Spontaneously Hypertensive Rat
Authors: Shanks Julia , Manou-Stathopoulou Sotiria , Lu Chieh-Ju , Li Dan , Paterson David J , Herring Neil
Published: June 2021

The spontaneously hypertensive rat (SHR) has increased peripheral cardiac sympathetic activity compared to Wistar Kyoto (WKY) controls. However, it is not clear whether this precedes the development of hypertension. We therefore compared peripheral cardiac sympathetic responsiveness in the SHR to the WKY at 4 weeks of age. Ventricular weight/body weight ratios and mean arterial pressure measured via the left carotid artery demonstrated that SHRs (n=8) were without left ventricular hypertrophy and were normotensive at this age compared to WKYs (n=7). In isolated cultured neurons from stellate ganglia, the magnitude of the calcium transient in response to high extracellular potassium depolarization was significantly (p<0.05) larger in SHRs (n=10) compared to WKYs (n=8). There was also a significantly greater release of [3H]‐norepinephrine (NE) to field stimulation (5Hz) of right atria (n=7 for both groups) and higher plasma levels of the sympathetic co‐transmitter neuropeptide Y (NPY) (n=12 for both groups). In an isolated organ bath atrial preparation with intact right stellate ganglion there was a greater heart rate response to right stellate stimulation at 5 and 7Hz in SHRs (n=9) compared to WKYs (n=7). Increased cardiac sympathetic neurotransmission may therefore represent an early marker in the development of hypertension driven by larger calcium transients.

Supported by the British Heart Foundation
The attenuation of the stress evoked tachycardia produced by angiotensin‐(1–7) in the basolateral amygdala is reversed by blockade of Mas receptor
Authors: Fontes Marco Antônio Peliky , de Figueiredo Muller Ribeiro Flávia Camargos , Xavier Carlos Henrique , dos Santos Robson Augusto Sousa , Oscar Charles Gonzaga
Published: June 2021

We have previously reported that nanoinjection of angiotensin‐(1–7) [Ang‐(1–7)] into the basolateral amygdala (BLA) attenuates the cardiovascular response evoked by acute stress in rats. In this study we evaluated the contribution of Mas receptor in the anti‐stress effect produced by Ang‐(1–7) in the BLA. Under tribromoethanol anesthesia (250 mg/Kg i.p.), bilateral guide cannulas were implanted targeting the BLA of Wistar rats (n=5–6) for nanoinjection of saline (100 nl), Ang‐(1–7) (25 pmol/100 nl) or Ang‐(1–7) + A‐779 (25pmol+50pmol, 100 nl). Four days later, arterial lines were implanted for cardiovascular recordings. After 24h, nanoinjections into the BLA were performed followed by air jet stress (10L/min‐10min). In the saline group, stress evoked increases in MAP and HR (137±25 bpm; 24±2 mmHg). The attenuation of the tachycardia produced by Ang‐(1–7) was reversed by A‐779 [(Ang‐(1–7) −5±13 bpm vs. A‐779+Ang‐(1–7) 114±17 bpm, P<0.001]. The same was observed for the pressor response [Ang‐(1–7) 2±2 mmHg vs. A‐779+Ang‐(1–7) 15±1 mmHg, P<0.05]. We observed in two rats that the attenuation of the tachycardic response produced by 5 sessions of stress conditioning was reversed by injection of A‐779 into BLA. These findings indicate that the Ang‐(1–7) may reduce the cardiovascular response to acute stress in the amygdala acting via Mas receptor. Support: CAPES, FAPEMIG, CNPq.
A2a adenosine receptors modulate cardiopulmonary chemoreflex control of regional sympathetic outputs via activation of GABAergic neurons within the caudal portion of the nucleus of the solitary tract (cNTS): functional and anatomical evidence
Authors: Minic Zeljka , Llewellyn-Smith Ida J , OˈLeary Donal S , Scislo Tadeusz J
Published: June 2021

The cardiopulmonary chemoreflex (CCR) may contribute to severe hypotension and sudden cardiac death when markedly activated. Since adenosine is released into the central nervous system during life threatening ischemia, we hypothesized that it may serve as a central negative feedback regulator for this reflex integrated in the cNTS. We have shown that CCR evoked sympathoinhibition was markedly attenuated after stimulation of A2a adenosine receptors in the cNTS (FASEB J 24: 624.11, 2010) and this attenuation was virtually abolished by GABAergic blockade (FASEB J 25: 844.3, 2011). These data imply that GABAergic neurons/terminals within cNTS express A2a receptors. We investigated this hypothesis using double immunofluorescent staining for A2a receptors (Cy3, red) and GAD67, the GABA synthetic enzyme (FITC, green). We observed extensive colocalization of A2a receptors with GABAergic neurons in the cNTS while we observed no such colocalization in more caudal portions of the medulla. These data support our functional findings that A2a adenosine receptors inhibit CCR responses by facilitating GABA release from nerve terminals in the cNTS. HL‐67814
Inhibition of AT1 receptors alters remodeling of the guinea pig cardiac plexus following chronic MI
Authors: Hardwick Jean C , Ryan Shannon E , Wilson Matthew J , Southerland E. Marie , Ardell Jeffrey L
Published: June 2021

Chronic heart disease induces remodeling in the guinea pig intrinsic cardiac plexus, which includes changes in responses to Angiotensin II (Ang II). We examined the role of Ang II in the remodeling process by using chronic treatment with either an AT1 antagonist (Losartan, 3mg/kg/day) or an ACE inhibitor (captopril, 3mg/kg/day) for 6 weeks following surgically‐induced myocardial infarction (MI). Intracellular voltage recordings from individual intracardiac neurons in whole mounts of the cardiac plexus were used to assess changes in physiological responses. Losartan and captopril treatments inhibited the synergistic effects of Ang II on the increase in neuronal excitability with either adrenergic or muscarinic agonists in MI, but did not alter the increase in excitability produced by adrenergic agonists alone. Losartan treatment increased synaptic efficacy for intracardiac neurons in MI animals; captopril did not. These results suggest that Ang II modulates neuronal remodeling post‐MI and that stimulation of AT1 receptors may be involved in the modulation of functional responses in the intrinsic cardiac plexus in the diseased heart.
Assessment of HD‐X11 Transmitter (DSI) for Telemetric Measurement of Arterial Pressure, Electrocardiogram, Heart Rate and Autonomic Function in Conscious Mice
Authors: Sabharwal Rasna , Chapleau Mark W
Published: June 2021

The small size of mice limits the number of variables that can be measured simultaneously by telemetry. The goal of this study was to evaluate the performance of the recently released HD‐X11 transmitter (DSI®) capable of recording blood pressure (BP), electrocardiogram (ECG), locomotor activity and body temperature. C57BL6 mice and mice deficient in the antioxidant enzyme methionine sulfoxide reductase (MsrA−/−) were studied before and during infusion (2wks) of angiotensin II (AngII) (1000 ng/kg/min). Measurements were compared with those obtained using the PA‐C10 transmitter capable of recording BP and activity only. Catheters/transmitters were chronically implanted under ketamine/xylazine anesthesia. Body weight, BP, heart rate (HR), and derived variables did not differ in mice implanted with HD‐X11 (n=4) vs. PA‐C10 (n=4) transmitters, before and during AngII infusion. Activity was lower in mice with the HD‐X11 probe (P<0.05). Both transmitters detected hypertension and decreased baroreflex sensitivity (BRS, sequence method) in MsrA−/− and AngII‐infused mice (Table). HR variability (SD) calculated from pulse intervals and R‐R intervals were strongly correlated (slope=1.17, r=0.97, P<.001), and reduced in MsrA−/− and AngII‐infused mice (P<0.05). We conclude: 1) HD‐X11 and PA‐C10 transmitters provide equivalent cardiovascular and autonomic data in normotensive and hypertensive mice; 2) Locomotor activity is lower in mice implanted with the HD‐X11, perhaps due to increased size/weight of the probe; and 3) Recording of both ECG and BP with the HD‐X11 can assure accurate measurements of HR variability and BRS, and define relationships between autonomic function and arrhythmias. (HL14388, DS1)

PA‐C10
HD‐X11

24hr BP (mmHg)
Baseline
AngII
Baseline
AngII

 C57BL6
106±1
147±11
105±3
144±3

 MsrA−/−
115±13
168±3
119±1
161±7

BRS(ms/mmHg)

 C57BL6
2.0±0.0
1.5±0.1
2.0±0.1
1.1±0.1

 MsrA−/−
1.1±0.0
0.7±0.2
1.0±0.1
0.6±0.1
Electroacupuncture Activated Pathway from Nucleus Tractus Solitarii to Rostal Ventrolateral Medulla
Authors: Guo Zhi-Ling , Longhurst John C.
Published: June 2021

Our previous studies have shown that electroacupuncture (EA) at the Jianshi‐Neiguan acupoints (P5‐P6, overlying the median nerve) attenuates sympathoexcitatory responses through its influence on neuronal activity in the rostral ventrolateral medulla (rVLM). The nucleus tractus solitarius (NTS) receives inputs from somatic nerves. Although there are direct projections from NTS to rVLM, it is unknown if these projections participate in central responses during somatic nerve stimulation. Moreover, there is no information on activation of NTS neurons by EA at P5‐P6 acupoints, which subsequently affects on the rVLM. Thus, the present study evaluated neuronal activation of the NTS in response to EA, with regard to their projections to the rVLM. Seven to ten days after unilateral microinjection of the retrograde tracer, rodamine‐conjugated microspheres (100 nl) into the rVLM, five rats were subjected to 30 min of EA at P5‐P6 acupoints applied bilaterally. Perikarya containing the microsphere tracer were found in the NTS of both groups. Compared to four controls (needle placement without electrical stimulation), c‐Fos immunoreactivity and neurons double‐labeled with c‐Fos, an immediate early gene and the tracer were more frequently found in the NTS of EA‐treated rats, particularly in the medial and lateral subdivisions of caudal and intermediate regions of the NTS extension. These results suggest that EA at P5‐P6 acupoints activates NTS neurons. Furthermore, EA‐activated NTS neurons frequently project directly to the rVLM, which is known to participate in EA‐modulation of sympathetic activity (supported by NIH grants HL‐72125 and HL‐63313).
Role of small conductance calcium‐activated potassium channels (SK2) expressed in hypothalamic PVN neurons in regulating sympathetic nerve activity (SNA) in rats
Authors: Gui Le , Gu Mingjun , LaGrange Lila P , Zhu Jianghua , Chen Qing-Hui
Published: June 2021

Recently, we reported that SK channels regulate membrane properties of RVLM projecting PVN neurons and that inhibition of SK channels increase in vitro excitability. Here, we determined in vivo the role of PVN SK channels in regulating SNA and mean arterial pressure (MAP). In anesthetized rats, bilateral PVN microinjection of SK channel blockade with apamin (0, 0.125, 1.25, 3.75, 12.5, 25 pmol; 50 nl) increased splanchnic SNA (SSNA), renal SNA (RSNA), and MAP in a dose‐dependent manner. Maximum increases in SNA and MAP elicited by apamin (12.5 pmol) in SSNA, RSNA and MAP were 330 ± 40% (n=8), 271 ± 40% (n=6), and 29 ± 4 mmHg (n=7), respectively. Pre‐treatment with DCEBIO (2.5 nmol), a SK channel opener, microinjected into the PVN significantly (p<0.05–0.001, n=5) diminished the sympathoexcitatory response evoked by apamin (102 ± 24% for SSNA, 78 ± 40% for RSNA and 16 ± 3 mmHg for MAP). DCEBIO alone was without effect on baseline SNA and MAP. Western‐blot examination of the PVN showed abundant protein expression of SK2 channels. Further immunohistochemistry studies showed that SK2 channels were primarily expressed in dorsal and lateral parvocellular divisions of the PVN. The majority of SK2 channel expression was localized in PVN‐RVLM neurons identified by retrograde labeling. We conclude that SK currents in the PVN inhibit ongoing SNA. Inhibition of SK2 channels may underlie the mechanism of increased SNA induced by apamin. Support: AHA 10SDG2640130 (QHC).
Acute hypernatremia increases the discharge of sympathetic‐vasomotor neurons in the rostral ventrolateral medulla
Authors: Stocker Sean D
Published: June 2021

Sympathetic‐regulatory neurons of the rostral ventrolateral medulla (RVLM) play a pivotal role in salt‐sensitive hypertension. Previous studies have demonstrated that several models of salt‐sensitive hypertension are associated with increases in plasma or cerebrospinal fluid sodium concentration. The purpose of the present study was to determine whether acute increases in plasma sodium concentration activate sympathetic‐vasomotor RVLM neurons. Extracellular single‐unit recordings were performed in urethane‐anesthetized, male Sprague‐Dawley rats (290–350 g). Intracarotid injection of hypertonic saline (250 mM NaCl, 100 uL over 10 s) significantly increased RVLM discharge in 83% (5/6) of neurons (4.1±1.4 to 9.6±2.9 Hz, P<0.01), elevatd renal SNA (38±10%, P<0.01), and increased arterial blood pressure (14±2 mmHg, P<0.01). These neurons were barosensitive as inflation of an aortic cuff increased arterial blood pressure (31±2 mmHg) but decreased RVLM neuronal discharge (4.2±1.4 to 1.5±0.5 Hz, P<0.01). All neurons were antidromically activated from the spinal cord and had an average conduction velocity of 3.5±0.8 m/s. Intracarotid injection of isotonic saline had no effect on any variable. These findings suggest that acute increases in plasma sodium concentration activate sympathetic‐regulatory neurons in the RVLM.
Behavioral assessments of mouse Strain differences in response to social stress
Authors: Jibitu Meskerem , Meyerhoff James , Chakraborty Nabarun , De Lima Thereza Christina Monteiro , Jett Marti , Hammamieh Rasha
Published: June 2021

In this study a mouse model is set up with a potentially unbiased platform to address the strain differences in response to social stress. Three different mouse strains of C57, BALB/c and DBA were submitted to 5 day and 10 day social stress (SS). Mice were kept inside an aggressor (AGG) home cage and randomly exposed to the AGG three times a day for one minute or for up to ten attacks in less than one minute, while control mice never experienced AGG proximity.

Behavioral assessments were performed using the partition/barrier test. Behaviors were recorded and analyzed using Etho Vision software. Subject mice were kept in the aggressor cage separated from the aggressor by a perforated divider.

Socially stressed mice persistently avoided the partition area that separated them from the aggressor mouse. C57 mice exhibited significant behavioral changes in response to stress. DBA strain mice showed lower susceptibility to SS than C57. When BABL/c mice were evaluated on the same scale of behavioral parameters, they showed intermediate levels of (SS) responses that were between the C57 and BALB/c responses.

Genomic and epigenetic studies are underway to understand these differences in stress response.
The Importance of Testes Function in Mouse Models of Cachexia
Authors: Hardee Justin P. , White James P. , Puppa Melissa J. , Aartun Johannes D. , Narsale Aditi A. , Sato Shu , Carson James A.
Published: June 2021

Hallmarks of cancer cachexia include chronic inflammation, insulin resistance and hypogonadism. Genetic and tumor implantation mouse models are methods used to study cancer cachexia. The purpose of this study was to explore the relationship between gonadal function and body weight (BW) loss in two mouse models of cachexia. Experiment 1: BALB/c mice were implanted with C26 tumor cells and monitored for 29 days. BW, testes weight, and circulating testosterone decreased in tumor bearing mice (p < 0.05). Experiment 2: Male ApcMin/+ (Min) mice were sacrificed at either 12, 14, or 20 weeks of age. Min BW, testes weight, and circulating testosterone decreased between 14 and 20 weeks of age (p <0.05). Testes expression of androgen receptor protein decreased, while Bax protein and pSTAT(Tyr705) increased between 14 and 20 weeks of age (p < 0.05). Testosterone levels and testes weight were correlated with BW loss in the Min mouse. Experiment 3: Male Min and C57BL/6 mice were treated with an IL‐6 receptor antibody or PBS control at 16 weeks of age for 2 weeks. Although cachexia decreased testes weight and circulating testosterone, IL‐6 receptor antibody administration attenuated these changes. In the C26 and Min mouse models of cachexia, decreased circulating testosterone appears to be an important regulatory event for the progression of the disease. Funded by NCI R01‐CA121249
Genetic Background (C57BL/6J Versus FVB/N) Influence in Global Gene Expression Profile to High Fat Diet
Authors: Christoffolete Marcelo Augusto , do Nascimento-Sales Michelle , Mendes Adriane Cecília Borges , da Silva Melo Suzane , Prata Rodrigo Ferreira , Ribeiro Miriam Oliveira
Published: June 2021

Genetic Background is a key player in metabolic phenotypes, however, gene modulation evoked by dietary interventions are still poorly understood.

Objective

Submit C57BL/6J (C57) and FVB/N (FVB) mice to high fat diet and determine pattern of gene expression.

Methods

Animals were on chow diet (16,8% protein, 73,5% carb and 4,8% fat) or High Fat Diet ‐ HFD (23% protein, 35,5% carb and 35,9% fat) for 68 days. At the end, animals were euthanized and tissues collected. Total RNA was extracted and submitted to microarray analysis.

Results

FVB mice gained ~5 and ~9g on chow and HFD, respectively. C57 mice gained ~0.5 and ~13g on chow and HFD, respectively. Liver shows ~700 genes up and ~900 genes down‐regulated in FVB compared to C57. HFD evokes up‐regulation of ~215 genes and ~200 genes while ~170 genes and ~240 genes were down‐regulated, in FVB and C57, respectively. WAT shows ~1050 genes up and ~1350 genes down‐regulated in FVB compared to C57. High Fat diet evokes in this tissue the up‐regulation of ~1900 and ~1300 genes while ~1200 and ~1000 genes were down‐regulated, in FVB and C57, respectively.

Conclusion

Liver gene expression is severely affected by genetic background.
GPR91 deletion improves lithium‐induced nephrogenic diabetes insipidus
Authors: Lam Lisa , Gevorgyan Haykanush A , Riquier-Brison Anne DM , Peti-Peterdi Janos
Published: June 2021

Lithium (Li) is a commonly used treatment for bipolar disorders. However, the use of Li is complicated by its renal side effect causing nephrogenic diabetes insipidus (NDI). The mechanism by which Li causes NDI is incompletely understood but cyclooxygenase 2 (COX‐2) and prostaglandin E2 (PGE2) are primary players. It has been established that Li inhibits luminal uptake of dicarboxylates including succinate resulting in their increased urinary excretion. Succinate is the ligand of the newly discovered metabolic receptor GPR91 that is highly expressed in the collecting ducts and its signaling involves COX‐2 and PGE2. Therefore, we tested the hypothesis that GPR91 plays an important role in Li‐induced NDI. Wild type (WT) and GPR91−/− mice (n=5 each) were fed normal and Li‐added diets for 14 days and then euthanized to collect kidney tissues. Li‐induced polyuria was significantly less in GPR91−/− mice (osmolality=604.85 ± 56.96 mOsm, urine output=10.96 ± 1.07 ml/24h) compared to WT mice (osmolality=394.60 ± 29.81 mOsm, urine output=19.37 ± 1.30 ml/24h) with no significant difference in Li intake. AQP2 protein abundance in the medulla was 1.5 fold higher in Li‐treated GPR91−/− versus WT mice. Also, urinary succinate excretion progressively increased during Li treatment. These data suggest that deletion of GPR91 leads to a significant resistance to Li‐induced polyuria and may provide a novel therapeutic strategy.
Perinatal taurine imbalance induces insulin resistance via the renin‐angiotensin system in adult female rats
Authors: Roysommuti Sanya , Thaeomor Atcharaporn , Jirakulsomchok Dusit , Wyss J. Michael
Published: June 2021

Perinatal taurine depletion followed by high sugar intake (post‐weaning) alters renin‐angiotensin system and glucoregulation in adult animals. This study tests the hypothesis that in adult female rats, perinatal taurine imbalance induces insulin resistance via the renin‐angiotensin system. Female Sprague‐Dawley rats were fed normal rat chow with 3% β‐alanine (taurine deficient, TD), 3% taurine (taurine supplemented, TS) or water alone (C) from conception to weaning. Their female offspring were then fed normal rat chow with 5% glucose in water (TDG, TSG, CG) or water alone (TDW, TSW, CW) throughout the experiment. At 7–8 weeks of age, all groups displayed similar body, heart, and kidney weights. Fasting blood sugar was not different among groups. Compared to CW and CG, perinatal taurine depletion but not supplementation slightly increased plasma insulin levels. High sugar intake slightly increased plasma insulin only in TSG. Surprisingly, captopril treatment significantly increased plasma insulin in all groups except CG, and the greatest was in TDG (>170% up). Changes in insulin sensitivity and insulin secretion paralleled the changes in plasma insulin levels. However, this insulin resistance did not lead to glucose intolerance or hyperglycemia. These data indicate that the renin‐angiotensin system attenuates glucose‐insulin dysregulation in adult female rats that are perinatally depleted of taurine.
Neuropeptide Y Immunoreactivity in Arterial and Venous Sympathetic Neurons
Authors: Shah Amit Harendra , Cano Georgina , Card J Patrick , Sved Alan F , Kreulen David L
Published: June 2021

Neuropeptide Y (NPY) is a co‐transmitter in postganglionic sympathetic neurons that can amplify the vasoconstrictor effects of norepinephrine. The ‘chemical coding’ hypothesis suggests that NPY is present only in vascular sympathetic neurons but this has not been studied using retrograde tracing of mesenteric blood vessel innervation. In the present study, two isogenic strains of pseudorabies virus (PRV) were used to retrogradely label postganglionic neurons in the celiac ganglion (CG) innervating mesenteric arteries and veins. Adjacent mesenteric arteries and veins of adult male rats were isolated and bathed with either PRV‐152 or PRV‐614. Fluoro‐Gold was injected i.p. prior to PRV exposure to identify postganglionic neurons. Of all the neurons counted in the CG, 16.9 + 5.5% were arterial neurons, 27.3 + 5.6% were venous neurons, 1.8 + 0.6% were both arterial and venous neurons, and 54.1 + 3.6% were unlabeled. However, only 34.8 + 2.2% of all arterial neurons were NPY‐immunoreactive and only 33.1 + 3.4% of all venous neurons were NPY‐immunoreactive. Thus, distinct populations of postganglionic neurons innervate arteries and veins. Also, we find that only approximately a third of sympathetic neurons innervating mesenteric vasculature were NPY‐immunoreactive.
CNS Gαi2subunit proteins – the origin of impaired fluid and electrolyte homeostasis and blood pressure regulation in a saltsensitive phenotype?
Authors: Wainford Richard David , Pascale Crissey L
Published: June 2021

Aim

We examined the role of hypothalamic paraventricular nucleus (PVN) Gαi2 proteins, which we have shown are upregulated to mediate renal sympathoinhibitory pathways to prevent body fluid imbalance and salt‐sensitive hypertension in Sprague‐Dawley rats, in the Dahl Salt‐Sensitive (DSS) vs. ‐Resistant (DSR) rat phenotypes.

Methods

DSR, DSS, or DSS rats receiving an i.c.v. infusion of a scrambled (S) or Gαi2 oligodeoxynucleotide (ODN; 25 μg/day) were fed a normal 0.4% (NS) or high 8% NaCl (HS) diet for 3‐ wks. On day 21 MAP (via DSI radiotelemetry probes), 24h metabolic balance, plasma norepinephrine (NE), plasma renin activity (PRA) & PVN Gαi2 protein levels were determined (N=6/gp).

Results

PVN Gαi2 protein levels significantly increased in response to HS intake in DSR but not DSS rats. In DSS rats CNS Gαi2 down‐regulation significantly exacerbated renal sodium retention, global sympathoexcitation & hypertension.

Conclusion

The failure to endogenously up‐regulate PVN Gαi2‐proteins in the DSS, but not DSR phenotype, is a likely origin of impaired fluid & electrolyte homeostasis and blood pressure regulation in the DSS rat. This is supported by increased dysregulation of sympathetically driven sodium retaining mechanisms and the exacerbated hypertension following CNS Gαi2‐protein down‐regulation in DSS rats ‐ HL107330.

DSR
DSS

NS
HS
NS
HS
HS+ S ODN
HS + Gαi2 ODN

MAP (mmHg)
122±3
126±3
129±4
161±4*
155±2*
178±3*≠

24h H2O balance (ml/day)
15±1
17±21
13±2
34±3*
32±2*
37±2*

24h Na+ balance (meq/day)
0.6±0.1
1.2±0.3
0.4±0.1
3.3±0.4*
3.5±0.2*
5.4±0.4*≠

PVN Gαi2 protein levels (normalized to GAPDH)
2.4±0.2
11.1±1.3*
0.64±0.1
0.54±0.1
0.8±0.1
0.1±0.1*≠

Index of salt‐sensitivity (slope)
4.8±0.4
0.6±0.3
0.55±0.2
0.3±0.1≠

Plasma NE (nmol/L)
49±3
36±2*
47±5
87±7*
84±8*
116±9*≠

PRA activity (Ang I generation ng/ml/h)
4.9±0.5
1.4±0.1*
5.2±0.5
1.7±0.2*
1.5±0.3*
1.6±0.4*

p<0.05 vs. respective NS gp,

p<0.05 vs. HS + S ODN gp.
Induction of non‐alcoholic fatty liver is prevented by betaine supplementation in rats
Authors: Ahn Chul Won , Kwon Do Young , Kim Sun Ju , Kim Young Chul
Published: June 2021

Previous work in this laboratory showed that betaine supplementation inhibited fat accumulation in liver of rats fed a high fat diet. In this study we examined the hepatoprotective mechanism of betaine against induction of non‐alcoholic fatty liver. Male rats were fed a high fat (HF) liquid diet for 3 wks. Betaine (1 % w/v) supplementation reduced the elevation of hepatic triglyceride (TG) significantly while increasing serum total cholesterol and TG levels. The decrease in phosphorylation of adenosine monophosphate‐activated protein kinase and acetyl‐CoA carboxylase in fatty liver was blocked by betaine supplementation. The elevated translocation of lipogenic transcription factors, sterol regulatory element binding protein‐1c and liver X receptor‐alpha, was normalized in the betaine‐treated rats. The HF diet intake resulted in reduction of betaine‐homocysteine methyltransferase expression and elevation of hepatic homocysteine, which was inhibited by betaine. Meanwhile, betaine supplementation increased hepatic apolipoprotein B protein expression, the obligatory platform for assembly of very‐low density lipoprotein (VLDL), and serum phosphatidylcholine, a component of VLDL particles. Taken together, the results suggest that the beneficial effects of betaine against hepatic steatosis may be accounted for by inhibition of lipid synthesis as well as by enhancement of its secretion.
Chitosan oligosaccharides ameliorate inflammation in two experimental models of colitis through inhibition of intestinal epithelial cell NF‐kB signaling and apoptosis
Authors: Muanprasat Chatchai , Yousef Mohammad , Pichyangkura Rath , Chatsudthipong Varanuj
Published: June 2021

Dysregulated mucosal immune response and intestinal epithelial barrier defect contribute to pathogenesis of inflammatory bowel disease (IBD). Chitosan oligosaccharide (COS), a biodegradation product of chitosan, has been recently shown to have anti‐inflammatory properties in vitro. We aimed to evaluate the therapeutic potential of COS in the treatment of IBD and to elucidate its mechanism of action. Anti‐inflammatory effects of COS were investigated both in mice and in human colonic epithelial cell line (T84 cells). In dextran sulphate sodium (DSS)‐induced colitis model, COS treatment significantly rescued dead rate and protected against inflammatory intestinal damage with the most effective dose being 10–20 mg/kg/day. In addition, COS treatment suppressed NF‐κB activation and decreased colonic tissue levels of TNF‐α and IL‐6. Similar protective effect was also observed in acetic acid induced colitis models. In T84 cells, COS suppressed NF‐κB activation and production of TNF‐α and IL‐6 under both LPS and TNF‐α‐stimulated conditions. Furthermore, TNF‐α and H2O2‐induced T84 apoptosis was significantly prevented by COS treatment. COS represents a promising therapeutic option in the treatment of IBD, which may preserve the intestinal epithelial barrier integrity and prevent initiation and progression of IBD.

This work was supported by the Thailand Research Fund and the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative.