bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibitors of aldose reductase and sorbitol dehydrogenase mitigate hyperglycemia‐induced arteriolar dysfunction
Authors: Veres Zoltan , Koller Akos
Published: June 2021

Hyperglycemia by activating the polyol pathway elicits increases in intracellular sorbitol and fructose concentrations. Previously we showed that in isolated arterioles administration of sorbitol (10−7 M) elicited oxidative stress, which interferes with nitric oxide (NO) bioavailability and thus dilations. We hypothesized that an elevated level of fructose also contributes to the development of hyperglycemia‐induced dysfunction of microvessels. In isolated rat gracilis muscle arterioles (~150 μm at 80mmHg), high glucose treatment (25 mM for 30 min) reduced flow dependent dilation (max: 39 ±2 vs. 15 ±1 %), which was significantly reversed by an aldose reductase inhibitor, zopolrestat (max: 27±2 %) and partially by a sorbitol dehydrogenase inhibitor (SDI), CP‐470,711 (max: 23±3 %). Also, SDI ‐ in part ‐ restored dilations to flow in the presence of elevated sorbitol. Incubation of arterioles with fructose (10−4 M) reduced flow dependent dilations (max: 39±2 vs. 12±2 %), which were not further affected by inhibition of NO synthase by L‐NAME, but were partially reversed by SOD/CAT and PGH2/TXA2 receptor blocker SQ‐29548. Thus, we suggest that hyperglycemia ‐ by elevating intracellular sorbitol and fructose levels ‐ induces oxidative stress, which interferes with NO‐mediated dilations and promotes PGH2/TXA2 release. Thus the polyol pathway may contribute to the development of microvascular dysfunction in diabetes mellitus.

(Grants: Hungarian SRF/OTKA T48376, K71591; HSC/ETT 364/2006 and AHA F. Aff. 0855910D, USA)
Historical Note: Yackzan of Vanderbilt, an unacknowledged investigator in the discovery of ergothioneine in biological tissues
Authors: Tipton Charles M.
Published: June 2021

In 1909, Tanret in France, extracted ergothioneine (ET) from the ergot of rye. Its empirical formula is C9 H15 O2 N3 ; regarded as a betaine, and recognized as the only naturally occurring thiolimidazole amino acid. Within its structure is the sulfhydryl group which is the main effector of antioxidation. Its biological importance is its role as an antioxidant; but, to have physiological significance, a transporter (ONCT1) is needed to enter the plasma cell. In 1950, it was known that ET was present in blood but unknown whether it was present in biological tissues. Kamal Yackzan, a graduate student in the laboratory of Dr. Oscar Touster at Vanderbilt University, investigated this subject by (a)altering concentrations and ratios of existing reagents of prevailing methods and (b) using paper chromatographic analytical techniques and found quantifiable concentrations were found in liver, kidney, cardiac and spleenic tissues. In 1951, these results were recorded in an obscure publication of the University (1). Three years later while using column chromatography, Melville confirmed Yackzan's tissue findings and that the liver and kidney had the highest concentrations with the heart and the spleen having the lowest values (JBC. 206: 221, 1954). In the decades that have followed,ET has received research and commercial interest as a strong antioxidant with therapeutic value for select health disorders and diseases.

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1. Yackzan, K. Paper chromatographic analysis for ergothioneine in tissue. In: Bulletin of Vanderbilt University Abstracts of Theses. Nashville, 1951, p. 56.
Decreased thermoregulatory precision contributes to the hypoxic decrease in body temperature in lizards
Authors: Cadena Viviana , Tattersall Glenn J
Published: June 2021

Low oxygen induces a decrease in body temperature (Tb) in most vertebrates. This response has been attributed to a decrease in the in the Tb set‐point, protecting organs from oxygen depletion. In addition, hypoxia decreases activity levels and, therefore, the propensity to move. In ectotherms, where thermoregulation is mainly behavioural, hypoxia is, thus, expected to impact the precision of thermoregulatory control. To determine if thermoregulatory precision is indeed decreased in hypoxia, we evaluated the variability and level of thermoregulation of bearded dragons at five oxygen levels in three different experimental settings: a dynamic temperature‐choice shuttle box, a constant temperature dual‐choice shuttle box, and a thermal gradient. A significant increase in the size of the Tb range was observed at the lowest oxygen level (4% O2) in the dynamic shuttle box, but not in the thermal gradient. This was accompanied by a Tb drop of 2‐ 4°C, the drop being greatest when Tb must be actively defended. Situations that force lizards to continually choose temperatures lead to an increase in Tb variability, which is further exaggerated in hypoxia. This study reveals that a decrease in thermoregulatory precision caused by a diminished propensity to move or effect appropriate thermoregulatory responses may partially explain the lowering of Tb observed in hypoxic ectotherms.
Enhanced angiotensin II‐induced aortic constriction in maternally separated rats is endothelium‐dependent and reactive oxygen species (ROS)‐independent.
Authors: Loria Analia Silvina , Kang Kyu‐ Tae , Pollock David M , Pollock Jennifer S
Published: June 2021

We previously have reported that Angiotensin II (AngII)‐induced constriction is enhanced in rats subjected to Maternal Separation (MS), a model of early life stress. The aim of this study was to investigate whether NO and/or ROS are involved in the increased Ang II‐induced constriction in MS rats. Male Wistar Kyoto rats were separated from their mother 3 hs/day from day 2 to 14 of life. Unseparated rats (MU) were used as control. Dose‐response curves to AngII (10−12 to 10−4.5 mol/L) were performed in endothelium‐intact and denuded thoracic aortic rings from MS and MU adult rats. NO basal tone was determined using a single dose of PE (10−8M, ±L‐NAME 100 μM). Inhibition of NOS in endothelium‐intact rings increased the % maximal vasoconstriction (% max VC) in MU rats (3.0±0.2 fold). After NOS inhibition, increase in % max VC was attenuated in MS rats (1.5±0.2 fold, p<0.05 vs. MU). Denuded rings showed similar % max VC in both groups. NO basal tone was reduced in MS rats (44.9±7.4 vs. 20.0±4.0, p<0.05). Inhibition of ROS with PEG‐SOD (1000 mU/L) or apocynin (300 μM) decreased the % max VC in MU rats (27.8±3.4 vs. 7.2±0.8 and 9.1±1.9, respectively, p<0.05). Either PEG‐SOD or apocynin did not modify the % max VC in MS rats. These data indicate that MS induces a dysfunction in Ang II mediated signaling in the endothelium, suggesting that early life stress mediates a pro‐contractile vascular phenotype.
O2, hypersalinity, and Cu2+ potentiate the toxicity of H2S in erythrocytes of a marine invertebrate
Authors: Ortega Jocelynn M , Kagan Courtney L , Julian David
Published: June 2021

Environmental stressors frequently occur in combination and may produce additive or antagonistic effects. In sulfidic environments, H2S exposure may be accompanied by hypoxia, a range of salinities and heavy metals. However, whether these stressors interact is unknown. We tested this using erythrocytes from the H2S‐tolerant marine polychaete Glycera dibranchiata. Erythrocytes were exposed for 24 h at 15 ?C to abiotic stressors simultaneously: H2S (0.03‐0.3%), anoxia, salinity (13‐51 ppt), and Cu2+ (33‐133 µM). Cell viability was measured using propidium iodide and calcein green AM labeling. Compared to control conditions (normoxia, 32 ppt salinity, and no H2S or Cu2+), 0.03% H2S did not change cell viability, but 0.3% H2S decreased cell viability by 71%. Anoxia alone and salinity alone did not decrease viability, whereas during H2S exposure cell viability was increased by anoxia and decreased by hypersalinity. Cu2+ at 33 µM decreased cell viability by 6%, but when combined with 0.03% H2S cell viability was reduced by 76%. Although the combination of anoxia, hypersalinity, and Cu2+, did not affect viability, all other combinations of three stressors showed a significant effect. These results suggest that the toxicity of H2S is increased by normoxia, hypersalinity, and Cu2+, indicating that realistic, complex environmental conditions must be considered when investigating H2S toxicity.
The Fast Skeletal Troponin Activator, CK‐2017357, Increases Skeletal Muscle Force in‐vitro and in‐situ
Authors: Russell Alan J , Lee Ken , Hartman Jim J , Marquez David , Hansen Richard , Muci Alex , Morgan Bradley , Jia Zhiheng , Malik Fady
Published: June 2021

CK‐2017357 is a member of a class of fast skeletal troponin activators that were identified by high throughput screening of skeletal sarcomere preparations. We sought to understand how this compound altered force development in isometric muscle fibers. Treatment of skinned rabbit psoas fibers with CK‐2017357 caused a dose‐dependent shift of the force‐pCa relationship without altering the Hill slope or maximum force, consistent with a calcium sensitizing effect on force production. In living rat FDB muscle, CK‐2017357 (10 μM) increased sub‐tetanic force (150% of control at 10 Hz) without altering maximum force. Similar experiments were performed using rat EDL muscle in‐situ, where nervous and vascular connections were left intact. Infusions of 10 mg/kg increased sub‐tetanic force by 200% at 30 Hz. In summary, we have identified a skeletal troponin activator that sensitizes the sarcomere to calcium and results in increased sub‐maximal muscle force development in‐vitro and in‐situ. We believe that this may translate to improved muscle function in diseases where muscle is compromised due to injury, disease or age.
Climbing exercise with a newly‐developed apparatus for hypertrophy of the FHL, FDS and FDP muscle in rats
Authors: Kondo Emi , Tai Shinji , Hirota Ayumi , Okada Sachiko , Imai Yoshitaka , Okamura Koji
Published: June 2021

The mass of the biceps brachii and the hind limb muscles, such as the gastrocnemius, do not significantly increase when performing climbing exercise with a newly‐developed apparatus in rats (EB 2007). The current study investigated whether the flexor hallucis longus (FHL), flexor digitorum superficialis (FDS) and flexor digitorum profundus (FDP), which contract to grip the wire mesh surface of the apparatus, increased in unloaded (Exp. 1) or weight‐loaded (Exp. 2) rats. In Exp. 1, 14 male rats were separated into a control (C) or exercise (E) group, group E was trained to climb inside a wire mesh box (1m height, 0.38 m width, 0.28 m depth) which was placed on a 53°C electric hot plate, without a weight. The exercise regimen consisted of 5 min × 6 sets/day, 3 days/wk for 8 weeks. Each group was pair‐weighed. In Exp. 2, the protocol was similar to Exp. 1 except that group E wore a rucksack containing a weight during the exercise. The masses of the FHL, FDS and FDP were significantly greater in group E than in group C in both Exp. 1 and Exp. 2 (P < 0.05). The differences in the FHL, FDS and FDP between groups E and C were 14, 13 and 17% in Exp. 1 and 12, 7 and 11% in Exp. 2, respectively, thus suggesting that the muscle hypertrophy in group E was not different between Exp. 1 and Exp. 2. In contrast, the gastrocnemius did not differ between the groups in either experiment. Therefore, the climbing exercise with the apparatus caused hypertrophy in the FHL, FDS and FDP but not in the gastrocnemius, thereby indicating that this exercise regimen causes regiospecific hypertrophy in the skeletal muscle. Although both models are considered to be useful as an exercise model of muscle hypertrophy since the hypertrophy was observed in both experiments, the unloaded model is considered to be a more user‐friendly model than the weight‐loaded model.
Evolutionary changes in myosin isoform expression in the tongue by quantitative PCR
Authors: Rahnert Jill A , Burkholder Thomas J , Sokoloff Alan J
Published: June 2021

Tongue and cranial muscles experience unique mechanical demands, particularly in the complex speech of humans, and may require specialized contractile properties. Cranial muscles, derived from somitomeres, display uncommon myosin heavy chain (MHC) isoforms, including cardiac‐α (Ca) and extraocular (EOM). Tongue muscles derive from somites and do not appear to express these isoforms by protein analysis. mRNA analysis may reveal expression of Ca and EOM. We hypothesize that MHC isoform expression will reflect the functional demands on the tongue, including expression of Ca and EOM. Expression of 8 MHC isoforms in intrinsic muscles of the tongues of human, monkey, and rat were determined by QPCR. Rats express mostly 2x (67 ± 4%) with substantial 2b (31 ± 4%) and no slow MHC. Humans and monkeys express primarily conventional MHCs: 2x (20 ± 6% human, 28± 6% monkey), 2a (50 ± 7%, 58 ± 7%) and slow MHC (24 ± 6%, 10 ± 2%). The expression of faster MHC isoforms in rat than primate tongue is similar to limb MHC protein expression patterns. Of the uncommon isoforms, Ca constitutes 5 ± 3% of total MHC in humans, and individual monkey samples contained up to 7.5% EOM MHC. Neonatal and embryonic isoforms contribute less than 1% of MHC in all 3 species. We conclude that the properties of conventional MHC isoforms may not provide sufficient functional diversity for tongue action. Supported by DC05017
Partial Pancreatectomized Diabetic Rats Present with Altered Skeletal Muscle Contractility and Phenotype
Authors: Gordon Carly S , Krause Matthew P , Cafarelli E , Hawke Thomas J , Riddell Michael C
Published: June 2021

People with long‐term type 1 diabetes (T1D) often have skeletal muscle complications (myopathy). Studies on T1D and skeletal muscle are limited and often use animal models that induce T1D with streptozotocin, which causes myopathy independent of hyperglycemia. The objective of this study was to characterize diabetic myopathy in a partially pancreatectomized (Px) model of T1D. Male Sprague Dawley rats were randomly assigned to either Px or sham surgery groups. Following 8 weeks of diabetes, Px had significantly lower body and gastrocnemius‐plantaris‐soleus (GPS) mass compared to shams (349.1±21 vs 484.4±10 g; 1.8±0.1 vs 3.3±0.1 g/g mass, respectively). In situ muscle stimulation of the GPS revealed a lower maximal force (Fmax) in Px vs shams (12.1±1.3N vs 22.6±1.8N, p<0.01), but was similar when corrected for muscle mass. During a 2 minute fatigue protocol, stimulating the GPS at ~50% Fmax (3 sec stimulation, 3 sec rest), Px had a lower rate of decline in force than shams (52.8±3.4% vs 69.7±7.2%, p<0.05). Histochemical analysis of the GPS demonstrates that Px have reduced myofiber area irrespective of fiber type and an increase in type IIa fiber number. These data suggest that sustained hyperglycemia/hypoinsulinemia induced by Px is characterized by changes in the skeletal muscle phenotype that result in compromised function. This research was funded by NSERC.
Resveratrol feeding may be therapeutic for dystrophic skeletal muscle
Authors: Selsby Joshua T. , Morine Kevin , Pendrak Klara , Tian Z. , Blanco Erica , Barton Elisabeth R , Sweeney H Lee
Published: June 2021

Increased PGC‐1α, either through a transgenic animal or gene transfer, provides therapeutic benefit to dystrophic skeletal muscle likely by increasing slow and oxidative protein expression. Resveratrol has been shown to activate SIRT‐1, a known deacetylase capable of acting on and increasing the activity of PGC‐1α. The purpose of this investigation was to determine if resveratrol supplementation could rescue dystrophic skeletal muscle. We hypothesized that daily feeding of resveratrol would improve muscle function in a similar fashion as PGC‐1α over‐expression. To test this hypothesis, 1‐mo old mdx mice were either fed a control diet (Con) or a diet containing resveratrol at 100 mg/kg/day (Res) for 8 wks. Following intervention, absolute muscle mass was reduced 18‐30% in the Res group compared to Con, however, body mass was also reduced in the Res group by 20% compared to Con. Relative muscle mass was similar between groups, except for the EDL, which was significantly smaller in the Res group. Solei in the Res group were more fatigue resistant than in the Con group, however, resistance to contraction induced injury was similar between groups. These data indicate that partial rescue of dystrophic pathology is possible through resveratrol. Perhaps, alternative sources or doses of resveratrol will provide a greater response. U54‐AR052646, F32AR055005‐01, AR053461, and PPMD.
Exercise attenuates doxorubicin‐induced skeletal muscle damage
Authors: Smuder Ashley J , Min Kisuk , Kavazis Andreas N
Published: June 2021

Doxorubicin (DOX) is an antitumor agent used in cancer treatment. However, DOX induces myocyte oxidative damage via increased free radical generation. In this regard, exercise presents a possible intervention for DOX toxicity through the induction of cytoprotective proteins. Therefore, we tested the hypothesis that DOX‐induced skeletal muscle damage could be attenuated by endurance exercise training. Sprague‐Dawley rats were randomly assigned to one of the following groups: sedentary, exercise, DOX, or exercise with DOX. Following the treatment protocol, soleus muscle was removed for subsequent analysis. Exercise prevented DOX induced increases in muscle lipid peroxidation. In addition, exercise training was sufficient in preventing muscle DOX induced HSP72 reduction. Furthermore, muscle levels of catalase, CuZn, and Mn‐superoxide dismutase increased with DOX suggesting antioxidant upregulation may act to combat increased oxidant production. Moreover, DOX resulted in increased muscle levels of ubiquitin‐protein conjugates as well as caspase‐3 and calpain I activity, all of which were attenuated with exercise training. These results suggest that DOX promotes skeletal muscle oxidative damage and protease activation. Importantly, exercise training appears to counteract the deleterious side effects of DOX.

Supported by an AHA grant awarded to ANK.
IL‐6 family members are sensitive to alcohol‐induced oxidant stress in rat plantaris muscles
Authors: Otis Jeffrey S , Guidot David M
Published: June 2021

Chronic alcohol abuse may produce oxidant stress and lead to muscle wasting and dysfunction. Our goals were to (1) minimize oxidant stress and attenuate plantaris atrophy by supplementing diets of alcohol‐fed rats with a glutathione (GSH) precursor, procysteine (PRO), and (2) identify the role of IL‐6 family members in alcoholic myopathy. After 35 weeks of daily alcohol ingestion, rats displayed plantaris atrophy, decreased GSH levels, decreased activities of GSH reductase and GSH peroxidase, and increased NADPH oxidase‐1 gene expression. Further, gene expressions of several catabolic factors were induced, including IL‐6, oncostatin M, atrogin‐1, and MuRF. Interestingly, 12 weeks of PRO supplementation in alcohol‐fed rats restored GSH levels and attenuated plantaris atrophy, but did not reduce other markers of oxidant stress or levels of these catabolic factors. Instead, PRO induced IGF‐1, and two anabolic IL‐6 family members, ciliary neurotrophic factor and cardiotrophin‐1. Together, these data suggest that PRO does not "turnoff" alcohol‐induced oxidant stress, but rather stimulates anabolic pathways to compete against these persistent catabolic factors. Supported by NIH/NIAAA (AA07190‐01A1) to JSO.
Subcontracture depolarization alters doublet potentiation
Authors: Barnes William Stephen
Published: June 2021

Doublet potentiation occurs when a brief, high‐frequency burst of pulses is added to a subtetanic train of pulses. The purpose of this investigation was to determine the effect of subcontracture K+‐induced depolarization on doublet potentiation. Sartorii muscles (60‐70 mg) from R. pipiens were excised and mounted in water‐jacketed (20°C) chambers containing oxygenated (100%) Ringer's solution (R; pH 7.2) containing 2.5 mM KCl (R+KCl2.5). Muscles were exposed to an 800 ms train (20 Hz) of supramaximal square‐wave pulses (.2 ms) via field stimulation. An additional pulse was inserted between the 9th and 10th pulses (ISI = 10 ms). Peak tensions (PT, g) for contractions (PTA‐E) between the eighth and twelveth pulses were determined. Tension‐time integrals (TTI, g·s) were calculated. This protocol was then repeated with the [KCl] of R increased incrementally up to 15 mM/L.

Subcontracture depolarization lessens the effect of transient high‐frequency paired stimulation and may mitigate its potential as a motor control strategy.
Angiotensin II is Necessary for Skeletal Muscle Regeneration Following Cardiotoxin‐induced Injury
Authors: Johnston Adam P.W. , De Lisio Michael , Bellamy Leeann , Parise Gianni
Published: June 2021

Angiotensin II (Ang II) is a pleiotropic peptide involved in vasoactivity as well as cellular growth and proliferation. We examined whether Ang II is also a mediator of muscle regeneration by administering an ACE inhibitor (captopril) following cardiotoxin (ctx)‐induced muscle injury in C57BL/6 mice. 10 days following ctx injection, captopril treated mice demonstrated a delayed response in the myogenic program evidenced by sustained expression of Pax7 and Myf5 transcripts while myogenin mRNA, a marker of differentiating myoblasts, was significantly inhibited. This delay was also evident at the myofibre level with captopril treated mice exhibiting a significant ~3.5 fold elevation in eMHC gene expression. Importantly, newly formed fibres in the captopril treated group demonstrated an impaired hypertrophic response as fibre cross‐sectional area (CSA) from day 10 to day 21 was increased by 81% in the control group and only 27% in the captopril group. This resulted in an overall difference of 25% in CSA between groups at day 21 post injury. These effects do not appear to be due to a deficiency in protein synthesis as myonuclear domain size was unchanged between groups. Rather, there appears to be a loss of satellite cell content as the captopril treated group demonstrated a ~29% decrease in Pax7+ cells by day 21 post injury. This research was supported by NSERC.
Effects of macrophage on unloading/reloading‐related regulation of muscle fiber size in op/op mice
Authors: Ohira Takashi , Wang Xiao Dong , Kawano Fuminori , Terada Masahiro , Yamamoto Hiroshi , Ito Tsunekata , Nakai Naoya , Goto Katsumasa , Ohira Yoshinobu
Published: June 2021

Roles of macrophages in the regulation of soleus muscle fibers of adult op/op (‐/‐) mice, without the macrophage colony‐stimulating factor, were studied. Mice were hindlimb‐suspended for 10 days and some mice were allowed to recover in the cage for additional 10 days. Macrophages, obtained from heterozygous () mice were transplanted into half of the recovery group i.p. immediately after termination of unloading. The samplings of soleus muscle were performed before suspension, immediately after suspension, and 10 days after recovery. The unloading‐related fiber atrophy in ‐/‐ mice without macrophage transplantation did not recover following 10 days of reloading, whereas that in the wild type and mice were increased to the control level. That in ‐/‐ mice was recovered by transplantation of macrophages. These morphological responses were closely related to the changes in the number of myonuclei and the quiescent and mitotic active satellite cells. It is suggested that the macrophages play one of the key roles in the regulation of muscle fiber size in mice.
Perinatal taurine status influences the effect of high sugar on renal potassium excretion in adult conscious female rats
Authors: Roysommuti Sanya , Lerdweeraphon Wichaporn , Jirakulsomchok Dusit , Wyss J. Michael
Published: June 2021

High sugar intake can impair renal function before increasing blood pressure. The present study tests the hypothesis that perinatal taurine status influences the effect of high sugar intake on renal potassium excretion in adult conscious female rats. Female Sprague‐Dawley rats were fed normal rat chow and given water alone (C), water with 3% beta‐alanine (taurine depletion, TD) or water with 3% taurine (taurine supplementation, TS) from conception until weaning. Their female offspring were treated with 5% glucose in drinking water (CG, TDG or TSG) or water alone (CW, TDW or TSW) from weaning onward. At 7‐8 weeks of age, renal potassium excretion at rest and after an acute normal saline load (5% of body weight) was tested in conscious, restrained rats. Perinatal taurine depletion, supplementation and glucose treatment alone did not alter renal potassium excretion at rest or after saline load. High sugar intake significantly decreased potassium excretion at rest in TDG and TSG (CW 2.1±0.4 μEq/min/gKW, CG 1.3±0.3 μEq/min/gKW, TDG 1.0±0.3 μEq/min/gKW, TSG 1.0±0.2 μEq/min/gKW; P < 0.05), but only the TDG group displayed decreased response to saline load. Tubular potassium reabsorption increased in TDG as indicated by decreased fractional potassium excretion. Plasma potassium levels significantly decreased only at rest in TDG. Glomerular filtration rates significantly decreased only at rest in TSW. Although non‐fasting blood sugar was higher in all glucose treated animals, their fasting blood sugar and glucose tolerance were not significantly different. The present data indicate that perinatal taurine status especially depletion heightens the adverse effect of high sugar intake on potassium excretion in adult conscious female rats.
The inhibitory effect of the glucagon‐like peptide‐1 analog exendin‐4 on NHE3 activity in proximal tubule cells is mediated by both PKA and EPAC signaling pathways
Authors: Carraro‐Lacroix Luciene Regina , de Oliveira‐Souza Maria , Girardi Adriana Castello Costa
Published: June 2021

Recent studies suggest a potential role for glucagon like peptide‐1 (GLP‐1) in salt and water homeostasis. Unpublished data from our laboratory indicate that exendin‐4, a GLP‐1 analog, reduces NHE3‐mediated Na/H exchange in the LLC‐PK1 cell line derived from kidney proximal tubule. The aim of this work was to define the signaling mechanisms involved in modulation of NHE3 by exendin‐4. Combination of 8‐Br‐cAMP and exendin‐4 does not result in an additive effect on the Na‐dependent intracellular pH recovery in LLC‐PK1 cells. The PKA antagonist H89 partially prevents inhibition of NHE3 activity by exendin‐4. No synergistic effect on the reduction of NHE3 activity is observed when LLC‐PK1 cells are treated simultaneously with exendin‐4 and cAMP analogs which selectively activate PKA or the exchange protein directly activated by cAMP (EPAC). Both exendin‐4 and PKA activation increase NHE3 phosphorylation levels by 70%, and this effect is completely abolished by pretreatment with H89. In contrast, EPAC activation does not alter NHE3 phosphorylation status. Collectively, these results suggest that exendin‐4 inhibition of NHE3 activity in LLC‐PK1 cells occurs through both PKA and EPAC‐dependent mechanisms.
Nitric oxide synthase and dynamin interactions in the renal inner medulla
Authors: Hyndman Kelly Anne , Musall Jacqueline B , Xue Jing , Pollock Jennifer
Published: June 2021

The movements of proteins in and out of the plasma membrane are important steps in regulating protein function. Dynamin (DNM) is a large GTPase that forms a helical collar around invaginated vesicles, and "pinches" it from the membrane during endocytosis. Recently, nitric oxide (NO) was reported to enhance DNM‐mediated endocyotosis through s‐nitrosylation of DNM in HEK cells, thus suggesting an important regulatory link between NO and endocytosis. We hypothesize that NO/DNM interactions are important mediators of ion channel function in the kidney. The purpose of this study was to determine which NOS and DNM isoforms directly interact in the renal inner medulla (IM). Sprague Dawley rats on a normal sodium diet were anesthetized and the IMs dissected and pooled per animal. These samples were immunoprecipitated with anti‐DNM and immunoblotted with NOS isoform specific antibodies. We determined there are direct interactions between DNM/NOS1 (alpha and beta splice variants), and DNM/NOS3 in vivo. In addition, DNMs1‐3 and NOS1 and NOS3 were immunolocalized to the IM collecting ducts (IMCD). We further confirmed our in vivo results by cotransfecting mIMCD‐3 and COS 7 cells with NOS1 and dynamin‐GFP constructs and determined these two proteins do directly interact in vitro. We conclude that DNM and NOS directly interact in the IMCD, leading us to further hypothesize that NO regulation of DNM is an important mechanism in regulating ion channel density in the IMCD.
The AP‐2 adaptin α/σ2 hemicomplex binds to a di‐isoleucine motif to mediate Kir2.3 endocytosis
Authors: Ortega Bernardo , Masson Amanda K , Welling Paul A
Published: June 2021

We have previously shown that a prototype basolateral membrane potassium channel, Kir2.3, contains a C‐terminal trafficking cassette comprised of a biosynthetic sorting signal, a PDZ binding site, and a novel di‐isoleucine endocytic signal. Present evidence indicates these signals sequentially interact with intracellular sorting machinery and Lin‐7/CASK to drive basolateral‐directed traffic in the biosynthetic and endocytotic pathways. Although channel endocytosis is dependent on the AP‐2 adaptor, it is unknown if and how the di‐isoleucine signal is recognized by the adaptin. To explore these questions, the subunits of AP‐1 and AP‐2 were expressed either individually or as hemicomplex forms and tested for interaction with Kir2.3 in a GST pull down assay. We found Kir2.3C binds strongly and preferentially to the AP‐2 α/σ 2 hemicomplex and modestly binds to the AP‐1γ/σ 1 hemicomplex. Kir2.3 did not recognize the β2/μ2 (AP‐2) hemicomplex. Replacement of the di‐isoleucine motif with di‐alanine significantly reduced the interaction. By contrast, the canonical dileucine motif binds preferentially to the γ/σ 1 (AP‐1) hemicomplex, with a weaker binding to α/σ 2 (AP‐2). In conclusion, the di‐isoleucine signal in Kir2.3 differs from canonical di‐leucine signals and may act purely as an endocytic signal, providing new insights into the trafficking mechanism of di‐hydrophobic sorting signals. Research supported by NIH.
Dynamics structures of mechanosensory cilia
Authors: Lo Shao , Przybylski Eric S , AbouAlaiwi Wissam A , Takahashi Maki , Nauli Surya M
Published: June 2021

Background

Non‐motile primary cilia have been proposed to be mechanosensory organelles in chondrocytes, cholangiocytes, osteocytes, renal epithelial, pancreatic epithelial, endocardial, and vascular endothelial cells. We report here the dynamic structure of the biophysical property of cilia.

Methods and Results

Using formvar as a flexible substrate, we record ciliary behavior for a period of time with a high resolution differential interference contrast microscopy. Under static conditions in the absence of fluid flow, we can observe that each well‐developed cilium contains a bulb in the middle of the ciliary shaft. In a less‐developed cilium, the bulb is less likely to be present. However, bending of cilium by mechanical fluid flow could induce the appearance of the bulb or bulbs within one cilium. Furthermore, fluid flow tends to dynamically move the bulb to the tip of the cilium.

Conclusion

We propose that the dynamics of ciliary bulb is mechanically regulated. Ciliary bulb may therefore have sensory function within mechano‐ciliary structure.
Vasopressin Regulation During Extracorporeal Membrane Oxygenation (ECMO) in a Pig Model of Septic Shock
Authors: Uyehara Catherine FT , Batts Sherreen G , Kinnison Martin W , McEntire Shane P , Sato Aileen K , Ichimura Wayne M , Hashiro Glenn M , Hernandez Claudia A
Published: June 2021

ECMO is used to provide cardiopulmonary support to critically ill neonates. Vasopressin (VP) plays a vital role in regulating mean arterial pressure (MAP) and regional blood flow in hypotensive disorders, but whether endogenous VP levels can be sustained during ECMO is unclear. In this study, 11 anesthetized, cardiac catheterized piglets were put on veno‐arterial ECMO either during control or endotoxin‐induced septic shock states. ECMO blood flow was delivered to maintain MAP at baseline levels. Plasma VP (pVP) was compared before and after stabilization on ECMO for 2 hours. ECMO caused pVP to drop by 30% (p<0.01) in both control and septic shock conditions. This drop may be due to the ECMO circuit adding to total circulating volume without additional release of VP. To test whether right atrial pressure (RAP) influence on VP release may be disrupted during ECMO due to partial cardiac bypass of blood flow, the relationship between pVP and hemodynamics during control was examined with stepwise regression. Pre‐ECMO, pVP negatively correlated with RAP and heart rate (r=0.83, p<0.05), but not MAP. In contrast, during ECMO, this pVP relationship with RAP was lost, but a strong positive correlation (r=0.76, p<0.05) between pVP and MAP became evident. Results indicate that during ECMO, low pressure atrial baroreceptors no longer regulate VP release, but VP control of MAP is maintained. Supported by US Army CRADA 0709‐T‐C454
Transient receptor potential vanilloid‐1 (TRPV1) and TRPV4 Null Mice Display Normal Salt Appetite
Authors: Taylor Anna Caitlynn , Stocker Sean D
Published: June 2021

The forebrain lamina terminalis plays an important role in body fluid homeostasis. Recent evidence suggests that both TRPV1 and TRPV4 channels contribute to central osmoregulation during acute and chronic sodium loading. The purpose of the present study was to determine whether TRPV1 and TRPV4 channels contribute to salt appetite. Wild‐type (WT) mice and mice lacking the TRPV1 and TRPV4 genes (TRPV1xV4‐/‐, n=4 per group) were fed a sodium‐deficient diet (0.01%) and given access to water and 0.3M NaCl for 10 days. Average 24‐h intake of water (2.66±0.16 vs 2.78±0.05mL) and 0.3 M NaCl (0.58±0.13mL vs 0.23±0.07mL) were not different between TRPV1xV4‐/‐ and WT mice. Hypovolemia, produced by sc injection of 20% polyethylene glycol (0.5mL) stimulated the ingestion of water and salt in both groups; however, TRPV1xV4‐/‐ and WT mice ingested similar amounts of water (1.63±0.06 vs 1.40±0.09mL, respectively) and 0.3 M NaCl (0.59±0.09 vs 0.54±0.10mL respectively) at 8 hrs. TRPV1xV4‐/‐ and WT mice drank similar amounts of water (0.15±0.03 vs 0.28±0.05mL) and 0.3 M NaCl (0.14±0.06 vs 0.15±0.05mL) after injection of saline vehicle (0.5mL). These findings indicate that TRPV1 and TRPV4 channels do not contribute to salt appetite. Supported by NIH HL090826, AHA 0630202N
Direct renin inhibition with Aliskiren lowers blood pressure and improves renal dysfunction in Ren2 rats
Authors: Nistala Ravi , Habibi Javad , Gutweiler Alex , Jellison Jessica , Rehmer Nathan , Hayden Melvin R , Sowers James R , Whaley‐Connell Adam
Published: June 2021

Renin has gained notoriety over recent times regarding its involvement and/or facilitation of pro‐inflammatory processes. Investigations into the mechanism of action of renin have revealed that this enzyme can not only activate the RAAS but can also act through the renin receptor directly, to affect kidney function. The transgenic Ren2 rat harbors stable copies of the mouse renin gene and has been well characterized to have hypertension, endothelial dysfunction and insulin resistance. Mouse renin transgene is expressed at high levels in the kidneys and is likely responsible for the pathophysiology that is so characteristic of this animal. We sought to reverse the pathology with a mouse specific direct renin inhibitor, Aliskiren (50mg/kg). We observed that Aliskiren lowered blood pressure significantly as expected (N=8, p<0.05). In addition, kidney oxidative stress, perivascular fibrosis and most importantly albuminuria were lowered in Ren2 transgenic animals (N=10, p<0.05). Interestingly, Aliskiren‐treated Ren2 rats demonstrated improved insulin signaling as measured with increase in total IRS‐1, decrease in phosphorylated Akt (thr308), and improved GLUT4 translocation to the plasma membrane fraction (N=5, p<0.05). Our data suggests that, Aliskiren treatment improves kidney function likely via inhibition of overactivity of RAAS and improved insulin signaling.
Disparate effects of chronic AT1 receptor blockade on blood pressure and renal injury in the aging female mRen2.Lewis strain
Authors: Yamaleyeva Liliya M , Lindsey Sarah H , Chappell Mark C
Published: June 2021

Salt‐sensitivity, a risk factor for cardiovascular and renal disease, increases with age or in women after menopause. Activation of the renin‐angiotensin system (RAS) may be an important pathogenic component of the salt‐sensitivite phenotype. In this study, we determined whether the angiotensin II receptor 1 (AT1R) antagonist losartan ameliorates blood pressure, proteinuria or vascular reactivity in the aging intact female mRen2.Lewis rats fed a high salt diet (HS, 8% NaCl). HS diet for 8 weeks starting at 60 wks of age increased systolic blood pressure [SBP: 150±7 vs.123±7 mmHg, p<0.05; n=5‐11], proteinuria [275±88 vs. 40±13 mg/kg/day, p<0.05], renal [p<0.05] and left ventricular [p<0.05] hypertrophy, without influencing creatinine clearance. Losartan treatment (20 mg/kg/day; 64 to 68 wks of age) had no effect on SBP and renal or cardiac hypertrophy, but significantly reduced proteinuria [62±28 mg/kg/day, p<0.05; n=8‐11]. Ang II‐dependent vasoconstriction in aortic rings from losartan‐treated animals was abolished, but the phenylephrine response was unchanged. Moreover, losartan treatment had no effect on acetylcholine‐induced relaxation. We conclude that in the aging salt‐sensitive female mRen2.Lewis rat, chronic RAS blockade does not reduce blood pressure, organ hypertrophy, or aortic vascular reactivity; however, AT1R antagonism is renoprotective.
Sinoaortic denervation prevents training‐induced plasticity in the central oxytocinergic (OT) system
Authors: Michelini Lisete C. , Cavalleri Marina T. , Ceroni Alexandre
Published: June 2021

Aerobic training (T) increase both ascending noradrenergic innervation to OT preautonomic neurons (Am J Physiol 2007;292:R1717) and OTmRNA expression within the hypothalamic paraventricular nucleus (PVN) (Hypertension 2005;46:1004), thus facilitating the slow down of the heart. We sought now to identify whether baroreceptor inputs affects T‐induced plasticity. WKY and SHR submitted to sinoaortic denervation (SAD) or SHAM surgery were trained (55% VO2 max, 5‐times/week, 1h/day) or kept sedentary (S) for 3 month. After pressure and heart rate (HR) measurement at rest, rats were anesthetized and perfused transcardiacally (0.01M PBS). Brains were removed, frozen and sliced to isolate dorsal brainstem (DBS, comprising the solitary‐vagal complex) and hypothalamic (directed to PVN) areas; tissue samples were processed for Real Time PCR. GAPDH was used as reporter gene. T‐induced pressure fall (‐8% in the SHR) and resting bradycardia (‐9% in both groups) were absent after SAD. SAD also completely blocked T‐induced OT mRNA expression in the PVN (4.3‐fold and 20% increases in SHAM WKYT and SHRT, respectively). Hypertension and SAD did not affect OT receptor expression within the DBS. In the chronic absence of baroreceptor inputs, the lack of plasticity on PVN OTergic neurons and HR response indicate that baroreceptor signaling plays a decisive role to drive beneficial T‐induced adjustments.

Support

FAPESP, CNPq.
Effect of aging and exercise training on eNOS uncoupling and O2‐ signaling in skeletal muscle arterioles
Authors: Sindler Amy Lynn , Delp Michael D , Reyes Rafael A , Wu Guoyao , Muller‐Delp Judy M
Published: June 2021

Reduced availability of tetrahydrobiopterin (BH4) contributes to the age‐related decline of nitric oxide (NO)‐mediated dilation in soleus muscle arterioles. Depending on the availability of substrate and/or necessary co‐factors, eNOS can generate NO and/or superoxide (O2−). We evaluated the effects of age and exercise on flow‐induced dilation (FID) and flow‐induced production (FIP) of NO and O2− in soleus muscle arterioles. Young (4 mo) and old (22 mo) male rats were exercise trained (ET) or remained sedentary (SED) for 10 wks. FID was studied in soleus muscle arterioles under control conditions and during the blockade of O2− and/or hydrogen peroxide. FIP of NO and O2− were determined. Arteriolar BH4 levels were also determined. ET enhanced FID in Y and O rats. ET restored BH4 and improved FIP of NO in arterioles O rats. FIP of O2− was higher in OSED vs. YSED. ET increased FIP of O2− in arterioles from Y rats. O2− scavenging with Tempol reduced FID in all rats except YSED. Addition of catalase to Tempol‐treated arterioles eliminated FID in all groups. Catalase alone reduced FID in all groups except OSED, with the greatest effect in YSED. FID was restored when deferoxamine, an iron chelator, was added to Tempol‐treated arterioles. These data indicate that uncoupling of eNOS contributes to the age‐related decline in FID; however, reactive oxygen species are required for FID in soleus muscle arterioles from Y and O rats.
Effect of acute inactivity on endothelin‐1‐mediated vasoconstriction in skeletal muscle feed arteries of diabetic rats
Authors: Larned Kristi J , Bender Shawn B , Laughlin M. Harold
Published: June 2021

Physical inactivity and type 2 diabetes (T2D) are independent cardiovascular risk factors. Little is known, however, concerning the effect of acute inactivity on vascular function in T2D. This understanding is important since many patients with T2D become sedentary. A common feature of T2D‐related vascular dysfunction is enhanced constriction to endothelin‐1 (ET‐1), a potent endothelium‐derived vasoconstrictor. Therefore, we examined the impact of acute inactivity on vascular reactivity to ET‐1 and endothelin type A (ETA) receptor expression in skeletal muscle feed arteries from the T2D OLETF rat. OLETF rats were housed with either no access to running wheels (SED) or open access for 16 wk. Inactivity was induced by locking the running wheels 5 hr (WL5) or 53 hr (WL53) prior to sacrifice. ET‐1‐mediated vasoconstriction of isolated, pressurized gastrocnemius feed arteries was enhanced two‐fold in SED T2D animals compared to non‐diabetic controls and this was unaffected by acute inactivity (WL5 and WL53). In addition, ETA receptor protein expression was not altered by acute inactivity in T2D feed arteries. Therefore, acute inactivity does not exacerbate vascular dysfunction via the ET‐1 system in the T2D skeletal muscle vasculature. Supported by NIH grant HL36088.
Exercise, but not perivascular fat, effects coronary artery vasomotor function in a familial hypercholesterolemic (FH) porcine atherosclerosis model
Authors: Bunker Aaron , Thorne Pam , Laughlin M. Harold
Published: June 2021

Our lab has shown that left circumflex coronary artery (LCX) PAT blunts endothelin‐1 (ET‐1) induced maximal (max) contractions in normal pigs on low and high fat diets. Other studies report that PAT exerts anti‐contractile effects on agonist‐induced arterial contraction via release of a relaxing factor that acts on the underlying vasculature. The purpose of this study was to test the hypotheses that: PAT blunts LCX contraction in FH pigs and that EX augments this anti‐contractile effect. Male FH pigs were divided into EX (n=13) and sedentary (SED) (n=15) groups. LCX reactivity to angiotensin II (AngII), bradykinin (BK), ET‐1, and sodium nitroprusside (SNP) was evaluated in vitro, with intact or removed PAT (+/‐PAT). LCX relaxation induced by BK and SNP was not altered by EX or ‐PAT. AngII‐induced max LCX contraction was nearly 2‐fold higher in EX than SED +PAT (p=0.1275). There was no difference between SED and EX responses to AngII ‐PAT rings. ET‐1 induced max contraction of LCX was not different between SED ‐PAT and SED +PAT, or between EX ‐PAT and EX +PAT. LCX from SED pigs exhibited a greater ET‐1 induced max contraction than LCX from EX pigs (p<0.05). We conclude that while PAT had no effect on LCX agonist‐induced contractions, EX did act to blunt ET‐1 induced max contractions in LCX +PAT and ‐PAT. (NIH HL‐52490)
Baroreflex gain and insulin sensitivity change in parallel during gestation in rats
Authors: Zhao Ding , Azar Afaf S. , Page Mollie , Brooks Virginia L.
Published: June 2021

Pregnancy (P) decreases baroreflex gain (BRG). Our recent studies in rabbits suggest that decreases in insulin sensitivity (IS) contribute to the BRG impairment: BRG falls in parallel with IS at end gestation, and treatment with the insulin sensitizing drug, rosiglitazone, normalizes BRG. In rats, IS falls at midgestation (days 11‐12 of 21 day gestation), rises to normal by day 15, and then decreases again just before delivery. However, whether BRG exhibits a similar pattern, or whether IS and BRG increase in parallel postpartum (PP), is unknown. To test these hypotheses, BRG was measured in conscious virgin (V) rats, during P [days 11‐12 (P12), 17‐18 (P18), and 19‐20 (P20)], and on PP days 3‐4 (PP3) from a 4 parameter sigmoidal fit of heart rate responses to slow (3‐5 min) ramp changes in arterial pressure. In addition, we measured IS using the hyperinsulinemic‐euglycemic clamp method in V, P20 and PP3 rats. Compared to V rats (4.5±0.5 bpm/mmHg, n=10), BRG was decreased (P<0.05) on P12 (2.8±0.1 bpm/mmHg, n=7) and P20 (2.3±0.4 bpm/mmHg, n=8); however, BRG on P18 (3.8±0.1 bpm/mmHg, n=5) and PP3 (5.9±0.2 bpm/mmHg, n=3) were not different from V. Similarly, IS was decreased on P20 (V, 32±2 mg/kg•min, n=6; P20, 14±1 mg/kg•min, n=3; P<0.05) but normalized by PP3 (30±2 mg/kg•min, n=8). In conclusion, BRG and IS change in parallel during and following rat gestation, suggesting a mechanistic link. Supported by AHA, NIH HL088552.
A Selective Inhibitory Effect of the Posterior Cerebellar Vermis on 10‐Hz Sympathetic Nerve Discharge
Authors: Barman Susan M , Gebber Gerard L
Published: June 2021

We compared the changes in inferior cardiac sympathetic nerve discharge (SND) and mean arterial pressure (MAP) produced by aspiration of portions of lobule IX and in some cases lobule X of the posterior cerebellar vermis with those produced by chemical inactivation (muscimol microinjection) or electrical stimulation of the same regions. The experiments were performed on 13 baroreceptor‐denervated and vagotomized cats anesthetized with urethane. Autospectral analysis was used to decompose SND into its frequency components. Special attention was paid to the question of whether the experimental procedures differentially affected the rhythmic and aperiodic components of SND. Aspiration or chemical inactivation with muscimol of lobule IX produced an approximately three‐fold increase in the 10‐Hz rhythmic component of SND (P = 0.05). In contrast, wideband SND at lower frequencies (= 6 Hz) was either slightly reduced or unaffected. Total power (0‐ to 20‐Hz band) was unchanged. Despite this, there was a statistically significant increase in MAP. High frequency (50‐Hz) electrical stimulation of depressor sites in lobule IX of the posterior vermis selectively and significantly reduced 10‐Hz power in SND. The lower frequency components of SND were unchanged. These results demonstrate for the first time a role of cerebellar cortical neurons of the posterior vermis in regulating the frequency composition of naturally occurring SND. The functional implications of these findings are discussed.

(Supported by NIH HL33266.)