bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytochrome B 5 reductase 3 regulates nitric oxide signaling at the myoendothelial by controlling hemoglobin redox cycling
Authors: Straub Adam , Lohman Alexander , Billaud Marie , Dwyer Scott , Best Angela , Gaston Benjamin , Isakson Brant
Published: June 2021

We have shown that hemoglobin subunit alpha (HBα) localizes to the myoendothelial junction(MEJ), the contact point between endothelial cells (EC) and smooth muscle cells (SMC) in small arteries to modulate nitric oxide (NO) scavenging, however, the redox mechanism controlling NO scavenging remains unknown. Since the iron (Fe) domain on Hb is critical for NO binding, we first tested the redox state of Fe in the thoracodorsal (TD) arteries and in isolated MEJ fractions from a vascular cell co‐culture (VCCC) model using UV‐visible absorption spectra, and found that Hbα resides primarily in Fe2+state. Using carbon monoxide to inhibit NO binding to Fe2+, we found reduced NO scavenging in TD arteries and in the VCCC. Since NO can react with Hb‐Fe2+ to yield Hb‐Fe3+, we tested if cytchrome B5 reductase 3 (CytB5R3), a known Hb‐Fe3+ reductase, is critical for Hb‐Fe3+ reduction. Western blot analysis showed CytB5R3 expression in TD arteries and in the VCCC. In addition, we found that Hbα co‐immunoprecipitates with CytB5R3. Using siRNA to knockdown CytB5R3 expression or propylthiouracil (PTU) to block its activity, we found attenuated NO scavenging in TD arteries and in the VCCC. Lastly, EC‐specific transfection of CytB5R3 or pretreatment with PTU inhibited phenylephrine‐mediated vasoconstriction. Together, these results suggest that CytB5R3 is critical for Hbα redox cycling to control NO scavenging and vasomotor tone.
Changes in central arterial stiffness during lower body negative pressure
Authors: Drury Taylor , Phillips Aaron A. , Bredin Shannon S.D. , Warburton Darren E.R.
Published: June 2021

In order to examine the influence of lower body negative pressure (LBNP) on central arterial stiffness, nine participants were exposed to consecutive 15 min stages of LBNP at increasing intensities (−20, −40, −60, and −80 mm Hg) until pre‐syncope. Carotid‐femoral pulse wave velocity (cPWV), and continuous measures of heart rate (HR), and systolic (SBP) and mean arterial blood pressure (MAP) were recorded at each stage. SBP low frequency band power (SBPLF) and the HRV low to high frequency band power ratio (LF/HF HRV) served as surrogate indicators of sympathovagal tone. T‐tests were used to compare changes in cPWV, HR, SBPLF, LF/HF HRV, and MAP from baseline to the maximum tolerated LBNP stage (LBNPmax). Delta scores (baseline to LBNP max) were calculated and Pearson correlation used to examine the relationship between cPWV and HR, SBPLF, or LF/HF HRV. cPWV, HR, SBPLF, and LF/HF HRV increased from baseline to LBNPmax (cPWV +294.07 ± 220.98, p = 0.004; HR +26.00 ± 17.34 bpm, p = 0.002; SBPLF +7.89 ± 9.94, p = 0.044; LF/HF/ HRV +6.62 ± 4.45, p = 0.002), while MAP did not change. cPWV was not significantly related to HR, SBPLF, or HF/LF HRV. In conclusion, it appears that central arterial stiffness increases during LBNP but is not related to HR, MAP, or indirect indicators of sympathovagal tone. Supported by the National Sciences and Engineering Research Council of Canada.
Neurotransmitter and neuropeptide control of the bone resistance vasculature
Authors: Prisby Rhonda , Campbell Jeremiah , Menezes Thomas , Kluess Heidi
Published: June 2021

Adequate bone blood flow is requisite for bone growth and repair. Regulation of bone blood vessels via the nervous system may provide a mechanism for the control of bone perfusion. We assessed whether the femoral principal nutrient artery (PNA), the primary conduit for blood flow to long bones, is controlled by various neurotransmitters and neuropeptides. Femoral PNAs from male Wistar rats (4 mon) were dissected and cannulated to assess vasomotor properties to increasing concentrations of neuropeptide Y (NPY; 0.125–1.000 mg/ml), Adenosine triphosphate (ATP; 0.78–50.00 μM/ml), mATP (4.95×10−4 – 4.95×10−8 M), norepinephrine (NE; 10−9–10−4 M), phenylephrine (PE; 10−9–10−4 M), clonidine (CLO; 10−9–10−6 M), vasoactive intestinal peptide (VIP; 10−10–10−4 M), calcitonin gene related peptide (CGrP; 10−10– 10−4 M) and Substance P (Sub P; 10−10–10−4 M). Interestingly, the femoral PNAs vasoconstricted to NPY, ATP and mATP (range = 31–57%). Additionally, vasoconstriction to NE and PE was ~77%, while CLO elicited only 33% vasoconstriction. Peak vasodilatations between 33–54% were observed to VIP, CGrP and Sub P. Data from the current study illustrate a potential role for these neurotransmitters and neuropeptides in the regulation of the bone resistance vasculature. Thus, the nervous system may provide a means of controlling bone blood flow during bone growth and repair. Supported by UTA REP grant 10466.
NO and O2•‐Interactions as a function of ratio of NO and O2•‐Production in the Microcirculation
Authors: Kar Saptarshi , Kavdia Mahendra
Published: June 2021

Oxidative and nitrosative stresses contribute to various pathological conditions in the vasculature such as lipid peroxidation, tyrosine nitration, cell necrosis and inflammation. The ratio of NO and O2•‐production is an important factor for the relative intensity of oxidative and nitrosative stress. Studies involving in vitro systems have shown that the maximum oxidative stress occurs when the ratio of NO and O2•‐production is equal to one. However, this behavior is not observed in biological systems. We developed a computational model to assess NO, O2•‐ and peroxynitrite levels as a function of O2•‐:NO production ratio in the microcirculation to understand how O2•‐:NO production ratio affects oxidative and nitrosative stresses. The key results include (a) increase in the production ratio results in a non‐linear decrease in maximum NO concentration and a linear increase in maximum O2•‐concentration and (b) at the production ratio of 1, both NO and O2•‐are not completely consumed that is observed in in vitro systems. The results show that for in‐vivo conditions such as microcirculation, oxidative stress can be significant even when production of O2•‐ is below or equal to the production rate of NO. Supported by NIH R01 HL084337.
Ankle position modifies tibialis anterior muscle perfusion and oxygenation in the human leg
Authors: Crater Ronald Brandon , Zhang Bing , Hargens Alan R.
Published: June 2021

Near‐infrared spectroscopy (NIRS) is a noninvasive technique used to monitor muscle oxygenation. Photoplethysmography (PPG) is a noninvasive measure of muscle microvascular blood flow. NIRS and PPG were used in seven tibialis anterior (TA) muscles of five healthy volunteers (3 male, 2 female) aged 31.2±7.8 years (mean, SD) in order to determine whether oxygenation and blood flow of the TA varied with ankle position. Data from the resting ankle position (mean of 11.5º plantarflexion) were compared to following test positions: full passive plantarflexion (PF), passive neutral (Neu) or 0º, and full passive dorsiflexion (DF). Mean oxygenation of the TA muscle yielded significant decreases for PF (p<0.01) and DF (p<0.01), but no significant difference for Neu (p=0.10). Mean perfusion of the TA muscle increased significantly for PF (p=0.027), Neu (p=0.012), and DF (p=0.010). Based on prior research, it was expected that oxygenation levels would decrease across the test ankle positions; however, it was not anticipated that perfusion would increase. Our novel results suggest that local compensatory vasodilation within muscle exists in order to increase microvascular blood flow under conditions of relative tissue hypoxia. These findings are important to the understanding of the local hemodynamic response in the human leg in relation to changes in ankle position from rest.
Exercise training and muscle microvascular oxygenation: role of nitric oxide bioavailability
Authors: Hirai Daniel M , Copp Steven W , Ferguson Scott K , Holdsworth Clark T , Musch Timothy I , Poole David C
Published: June 2021

Exercise training induces multiple adaptations within skeletal muscle that may improve local O2 delivery‐utilization matching (i.e., microvascular O2 pressure; PO2mv). We tested the hypothesis that increased nitric oxide (NO) bioavailability partly underlies improved muscle PO2mv kinetics from rest to contractions after exercise training. Rats were assigned to sedentary (S; n=18) or treadmill exercise trained (ET; n=10; 60 min/d, 5 d/wk, ~8 wks, 35 m/min; ‐14% grade) groups. PO2mv was measured via phosphorescence quenching in the spinotrapezius muscle at rest and during 1 Hz twitch contractions under control (CON, Krebs‐ Henseleit), SNP (NO donor; 300 μM) and L‐NAME (NO synthase blockade; 1.5 mM) superfusion. ET rats had greater VO2peak than S rats (S: 72 ± 2; ET: 81 ± 1 ml/kg/min; p<0.05). The rate of PO2mv fall during contractions was defined as ΔPO2mv/τ (where τ = time constant) and expressed in mmHg/s. Exercise training slowed ΔPO2mv/τ during CON (S: 1.46 ± 0.14; ET: 0.96 ± 0.14; p<0.05). Compared to CON, SNP slowed ΔPO2mv/τ only in S rats (S: 0.34 ± 0.06; ET: 0.65 ± 0.17; p<0.05) whereas L‐NAME speeded ΔPO2mv/τ only in ET rats (S: −12%; ET: 34%; p<0.05). Exercise training leads to greater microvascular oxygenation at the onset of contractions partly via increased NO bioavailability which likely constitutes an important mechanism for training‐induced metabolic adaptations.

(AHA Midwest Affiliate, NIH HL‐108328)
MMP2 deficient mice are protected from hydronephrosis after unilateral urethral obstruction
Authors: Tveitarås Maria Kathrine , Skogstrand Trude , Helle Frank , Leh Sabine , Reed Rolf K , Chatziantoniou Christos , Hultström Michael
Published: June 2021

Matrix Metalloprotease 2 (Mmp2) is a collagenase known to be important in the development of renal fibrosis. In unilateral urethral obstruction the obstructed kidney (UUO) quickly develops fibrosis, while the contralateral (CL) does not. The fibrotic process in Mmp2 deficient mice (KO) was investigated using heterozygote (HZ) and wild‐type mice as controls (WT).

UUO caused hydronephrosis, dilation of renal tubules, loss of parenchymal thickness, and fibrosis after one week. Sirius red quantification of fibrous collagen showed 0.13±0.03% positivity in CL and 0.33±0.05% in UUO (p<0.05). KO‐UUO developed less severe hydronephrosis and had preserved renal parenchyma.

Out of 48 genes quantified by PCR 26 were differentially expressed by ANOVA (p<0.05). 25 genes were up‐regulated in UUO wild‐type mice: Adamts1, ‐2, Col1a1, ‐2, ‐3a1, ‐4a1, ‐5a1, ‐ 5a2, Dcn, Fbln1, ‐5, Fmod, Fn1, Itga2, Loxl1, Mgp, Mmp2, ‐3, Nid1, Pdgfb, Spp1, Tgfb1, Timp2, Trf, Vim. Only 18 genes were differentially expression in KO‐UUO. Mmp9 was up regulated, while Mmp2, Fn1, Loxl1, Col5a1, Fbln5, Fmod, Fbln1 and Col5a2 were not changed in KO‐UUO.

In conclusion, Mmp2 KO protects against hydronephrosis after UUO and shows milder changes in gene expression compared to WT.

The study was supported by the Western Health Authority of Norway, Magn. Bergvall foundation and Lars Hiertas Minne Foundation.
Dysfunction of TRPV4 channels in the collecting duct‐derived cysts of ARPKD
Authors: Zaika Oleg L , Mamenko Mykola , Berrout Jonathan , OˈNeil Roger G , Pochynyuk Oleh
Published: June 2021

Collecting duct (CD) cells adjust water‐electrolyte transport in response to mechanical shear stress. The signaling cascade involves elevations of [Ca2+]i due to activation of the mechano‐sensitive Ca2+‐permeable TRPV4 channels. Impaired mechano‐sensitivity in the CD has been implemented in development of autosomal recessive polycystic kidney disease (ARPKD). Here, we combined direct measurements of [Ca2+]i with IHC in freshly isolated split‐opened collecting ducts of S/D rats and monolayer cyst fragments of the rat model of ARPKD, PCK 453, to explore a role of TRPV4 in patho‐physiology of the disease. Elevated flow increased [Ca2+]i in TRPV4‐dependent manner in individual cells of the CD from S/D rats but not in cyst cells from PCK 453 rats. Unexpectedly, prominent apical localization of TRPV4 was detected with IHC in both split‐opened CD and CD‐derived cysts. Pharmacological stimulation of TRPV4 with highly potent activator, GSK1016790A elicits 4 times greater elevations in [Ca2+]i in normal CD than in cyst cells. Consistently, ATP‐mediated activation of TRPV4 was virtually absent in freshly isolated cyst segments from PCK 453 rats. Therefore, we have concluded that the development of ARPKD, whilst does not alter cellular localization, leads to functional impairment of TRPV4 contributing to disrupted mechano‐sensitivity in the CD‐derived cysts.
Low dose PPARγ agonist inhibition of cyst growth in the PCK rat model of polycystic kidney disease
Authors: Flaig Stephanie Marge , Carr Alexander , Gattone Vincent , Blazer-Yost Bonnie
Published: June 2021

Polycystic kidney diseases (PKD) are genetic disorders characterized by fluid‐filled cysts in kidney tubules and liver bile ducts with fibrosis. Cyst enlargement is due, at least in part, to Clsecretion via the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Our previous studies demonstrated that PPARγ agonists, insulin sensitizing drugs used to treat diabetes, inhibit Cl− secretion by collecting duct principal cells via decreased CFTR synthesis. The dose response curves for Cltransport paralleled the EC50's for receptor transactivation with a leftward shift, suggesting an increased sensitivity for inhibition of Cl− secretion. Our previous preclinical studies showed that high (20 mg/kg BW) dose pioglitazone, a PPARγ agonist, inhibited cyst growth in the PCK rat model of PKD. The current study was designed to show a class action of PPARγ agonists and to determine if low doses of rosiglitazone, another PPARγ agonist, inhibit cyst growth and avoid adverse side effects. A 24 week feeding study using 3 doses of rosiglitazone (4, 0.4, 0.04 mg/kg BW) showed that the lowest dose (0.04 mg/kg BW) significantly slowed renal cyst growth. Funding: CTSI ‐ IU School of Medicine.
Diuretic Vs toxic effects of Aerva lanata (Polpala) on kidney of Sprague Dawley rats
Authors: Gunatilake Mangala , Lokuhetty Dilani , Herath HMDR , Edirisuriye DT , Bartholameuz NA , Wijayabandara J , Kularatne MU , Anand Date
Published: June 2021

Experimental studies were carried out in two phases to evaluate diuretic effect of the herb using hydrated rat assay technique and to determine possible toxic effects of prolonged use of Aerva lanata on the urinary tract of rats. Phase I – Two different concentrations of extract prepared using fresh and dried forms Aerva lanata was administered orally to 24 healthy rats. Findings of test groups were compared with those from rats administered water (negative control) and frusemide (positive control). Phase II – Two different concentrations of dried infusion of Aerva lanata was administered orally to 40 healthy rats for 30 days as per the WHO guidelines. Control group of rats were given water. Creatinine clearance (Ccr) and urine flow rate (UFR) were calculated to determine the effect of the herb on renal function. Kidneys, bladders and ureters were harvested for light (LM) and electron microscopic (EM) studies. Crude aqueous plant extract showed a diuretic activity. Ccr and UFR of test groups were not statistically different when compared with that of control group. LM of kidneys, ureters and bladders were negative for toxic changes. EM of kidneys showed significant changes in proximal convoluted tubular epithelial cells of rats in test groups. EM changes indicate that long term use produces structural changes.
Exercise training alters postprandial serum RBP4 in older adults
Authors: Brandauer Josef , Weiss Edward Paul , Berardinelli Danielle Marie , Hagberg James Michael
Published: June 2021

Serum retinol binding protein 4 (sRBP4) is a specific carrier for retinol and an adipokine that has been associated with insulin resistance. The purpose of this study was to determine the effect of six months of endurance training on fasting and postprandial sRBP4 concentrations in response to ingestion of a high‐fat, high‐calorie meal. Sedentary 50‐ to 75‐year‐old men and women (n=11) underwent endurance training (≈70% VO2 max, 20–40 min/session, 3–4 times/week). Serum samples were obtained after an overnight fast and periodically for 4 hours after the ingestion of a high‐fat meal. Serum RBP4 was assayed via immunoblotting. Fasting sRBP4 levels remained unchanged following endurance training (1.00±0.107 (mean±S.E.M) arbitrary units [AU] vs. 0.99±0.138 AU; p=0.93). A postprandial sRBP4 peak was observed at 60‐min, which was absent in the trained state (sedentary, +0.19 ±0.097 AU; trained, −0.10 ± 0.049 AU, p=0.01). Training had a prolonged effect on postprandial sRBP4 as assessed via incremental area under the curve (AUC) calculations (sedentary, 17.5±11.87 AU·180 min−1, trained, −9.7±5.07 AU·180 min−1; p=0.03). In conclusion, fasting sRBP4 remained unchanged after six months of endurance training. The postprandial serum RBP4 increase observed in sedentary individuals was attenuated following exercise training.
HDAC6 regulates mitochondrial oxidative phosphorylation by ATP synthase beta subunit acetylation in diabetic cardiomyopathy
Authors: Jagannathan Rajaganapathi , Baseler Walter A , Thapa Dharendra , Croston Tara L , Shepherd Danielle L , Nichols Cody E , Hollander John M
Published: June 2021

Diabetes‐induced cardiac mitochondrial dysfunction has been implicated as a trigger for pathogenesis in cardiac failure. Cardiac mitochondria consist of two distinct subpopulations, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). Our present work demonstrated that type 1 diabetic cardiac mitochondrial subpopulations have an abundance of acetylated proteins. We identified by mass spectrometry, 11 proteins that are acetylated in cardiac mitochondria of type 1 diabetic mice. ATP synthase beta subunit (ATP5B) was hyperacetylated in diabetic heart. For the first time we identified HDAC6 as a novel deactylator of mitochondrial proteins. HDAC6 contains an N‐terminal mitochondria targeting sequence which was confirmed by mass spectrometry. Cardiac mitochondrial basal levels of HDAC6 showed prominent differences between subpopulations. Further, diabetes‐induced cardiac mitochondrial HDAC6 expression is ablated in both subpopulations however IFM HDAC6 levels are more dramatically decreased. We observed a significantly increased level of acetyled ATP5B and decreased HDAC6 association in diabetic IFM. These findings identify acetylation as a novel regulatory mechanism for mitochondrial oxidative phosphorylation and that HDAC6 modulates mitochondrial intermediary metabolic molecules during the pathogenesis of diabetic cardiomyopathy. (Support: NIH DP2‐ DK083095)
Homocysteine levels and outside‐in insulin signaling in human and mouse tissues
Authors: Pansuria Meghana , Yang Xiao-Feng , Wang Hong
Published: June 2021

Hyperhomocysteinemia (HHcy) and insulin resistance both are associated with the atherosclerotic cardiovascular disease. However, the effect and mechanism of homocysteine (Hcy) metabolism on insulin signaling genes has not been studied. We examined the expression profiles of insulin signaling genes involved in PI3K and MAPK pathway through database mining approach. Furthermore, we analyzed the relationship between mRNA levels of insulin signaling genes, tissue Hcy, S‐adenosyl homocysteine (SAH), and S‐adenosyl methionine (SAM)/SAH ratio in mouse tissues. We found PI3K pathway genes are differentially expressed in both human and mouse tissues, with humans having the high expression of PI3K pathway genes in classic insulin sensitive tissues (muscle, liver, and adipose tissue). This suggests that humans have tissue specific regulation of insulin signaling genes involved in PI3K pathway. Moreover, PI3K pathway genes PDK1, Akt1, RPS6KB1, and HSL were negatively correlated with tissue Hcy and SAH levels. In contrast, MAPK pathway genes were mostly ubiquitously expressed in human tissues examined. MAPK pathway genes Grb2, MAP3K2, and Erk2 were negatively correlated with tissue Hcy and SAH levels. Our study indicates that Hcy metabolism is negatively correlated with insulin signaling genes. HHcy may impact on obesity and type 2 diabetes via suppression of insulin signaling. Our work is supported by NIH grant.
Duration of fast alters fasting glucose levels, response to oral glucose load, and weight in the mouse
Authors: Schork Pauline , Johnson Kathryn M.S.
Published: June 2021

Fasting alters glucose metabolism and insulin resistance. To determine the effect of various fast durations on circulating glucose levels prior to and after an oral glucose load, C56BL/6 mice were fasted for 0, 5, 10, or 24 hours (n =3–4 per group). After a 30‐min baseline period, mice received an oral glucose load (2 g/kg, post‐fast body weight) via gavage. Blood glucose levels were measured during baseline and 15, 30, 45, 60, 75, and 90 min after oral glucose administration. Post‐fast body weight was significantly lower after a 24‐hour fast (20.6 ± 0.6 g), compared to 0, 5, or 10 hours of fasting (24.0 ± 0.5, 22.7 ± 0.6, and 23.1 ± 0.7 g, respectively; all p ≤ 0.01). Fasting blood glucose levels were not different among the 0‐, 5‐, and 10‐hour fasted groups (173 ± 8, 198 ± 2, 206 ± 10 mg/dL, respectively); however, after a 24‐hour fast, baseline levels were significantly lower (120 ± 20 mg/dL; all p < 0.01). Mean increase from baseline blood glucose levels following oral glucose administration were significantly greater in the 5‐hour fasted group (303 ± 10 mg/dL) than either the 10‐ or 24‐hour fasted animals (253 ± 14 and 254 ± 20 mg/dL, respectively; both p = 0.01). These results indicate that fast alters glycemic parameters in mice; therefore, it is not appropriate to compare metabolic finding in mice fasted for different durations. P. Schork was supported by The American Physiological Society Frontiers in Physiology Fellowship.
Overexpression of Na/Ca exchanger‐1 (NCX1) in vascular smooth muscle leads to elevated basal blood pressure and increased responses to Angiotensin II
Authors: Chen Ling , Li Meng , Wier W. Gil , Wang Jingyu , Zhang Jin
Published: June 2021

Ca2+ entry via vascular smooth muscle (VSM) Na+/Ca2+ exchanger type‐1 (NCX1) was reported to trigger salt‐dependent hypertension. Here, we investigated whether VSM‐specific NCX1 overexpression modifies basal blood pressure (BP) and Angiotensin II (ANG)‐induced BP response. Adult wild type (WT) and age‐matched transgenic mice that express ~7 fold higher VSM NCX1 protein (NCX TG) were used. Under 1.5% isoflurane anesthesia, BP was continuously recorded with a Millar catheter via right carotid artery. NCX TG mice had significantly higher baseline BP compared to WT controls (95 ± 9 vs 81 ± 2 mm Hg, P < 0.05, n = 6 and 8). ANG injection (30 ng/kg in 50 μl saline, 10s, i.v.) caused a significantly greater BP elevation in NCX TG than in WT mice (33 ± 3% vs 20 ± 3%, P < 0.05; n = 3 and 6). A higher dosage of ANG (300 ng/kg) resulted in greater BP elevation in both groups, but no significant difference was observed between genotypes (38 ± 4% vs 52 ± 6%; P = 0.076). Thus, VSM NCX1 overexpression increases basal BP and exaggerates acute BP response to ANG. The elevated basal BP in NCX TG mice indicates that arterial NCX1 normally provides net Ca influx in the basal state. In this case, both cytosolic and sarcoplasmic reticulum [Ca2+] would be expected to be elevated, providing both increased vascular tone in the basal state and increased vasoconstriction in response to G‐protein coupled receptor agonists, such as ANG. (Supported by NHLBI and AHA).
PPAR‐α regulates NOX2 and SOD expression in the heart during Angiotensin II hypertension
Authors: Wilson Justin L , Sankavaram Kavitha , Duan Rong , Allard Joanne S , Lee Dexter L
Published: June 2021

Previous reports suggest peroxisome proliferator‐activated receptor‐α (PPAR‐α) agonist attenuates hypertension by suppressing oxidative stress and increasing superoxide dismutase (SOD) activity. We tested the hypothesis that the absence of PPAR‐α would increase NOX2 and decrease SOD expression in mice hearts during Angiotensin II (ANG II) hypertension. Ten to twelve week old male PPAR‐α knockout (KO) mice and their wild‐type (WT) controls were implanted with biotelemetry devices and infused with ANG II (400 ng/kg/min) for twelve days. Separate groups of PPAR‐α KO and WT mice were treated with ANG II plus apocynin (1 g/1L) and ANG II + fenofibrate (500 mg/kg/day). Mean arterial pressure (MAP) and heart rates (HR) were similar between PPAR‐α KO and WT mice during the control period. On day twelve of ANG II treatment, MAP was significantly higher in PPAR‐α KO than WT, 161 ± 5 and 145 ± 4 mmHG, respectively. Fenofibrate significantly reduced MAP in ANG II treated WT mice (134 ± 5 mmHg) and apocynin reduced MAP in both ANG II treated PPAR‐α KO and WT mice. NOX2 expression was significantly increased in ANG II‐treated PPAR‐α KO mice hearts with no significant changes in ANG II‐treated WT mice. Apocynin significantly reduced NOX2 expression in ANG II‐treated PPAR‐α KO mice with no changes in WT. SOD expression in ANG II + fenofibrate was significantly higher than ANG II‐treated WT mice, with no differences between ANG II + fenofibrate and ANG II‐treated PPAR‐α KO. Our results suggest that increased NOX2 and decreased SOD expression in PPAR‐α KO mice hearts contributes to the significant increases in MAP during ANG II hypertension.
NALP3 Inflammasome Activation in the Coronary Arterial Wall of Obese Mice
Authors: Xia Min , Boini Krishna , Abais Justine , Li Xiaoxue , Xu Ming , Li Pin-Lan
Published: June 2021

Inflammasomes as intracellular inflammatory machinery has been reported to switch on the inflammatory response to various danger signals. The present study hypothesized that activation of NALP3 inflammasomes turns on the endothelial inflammatory response and results in arterial endothelial dysfunction during the early stage of obesity and in response to adipokines. To test this hypothesis, we first observed increased inflammasomes activation in coronary arterial wall of obese mice, as shown by colocalization of NALP3 with apoptosis‐associated speck‐like protein (ASC) or caspase‐1 under confocal microscopy. The further studies in these obese mice found that plasma visfatin concentrations increased by 53.8% compared to normal mice. In addition, local transfection of visfatin shRNA in heart or daily injection of caspase‐1 inhibitor, WEHD (1 mg/kg/d) had no effects on visfatin level, but almost blocked obesity‐associated formation of NALP3 inflammasomes as shown by no co‐localization of NALP3 with ASC or caspase‐1. Correspondingly, IHC anlysis revealed that accumulation of T‐cells was abolished by visfatin shRNA and WEHD as shown by much less CD43 stains. In cultured mouse endothelial cells (ECs), confocal microscopy and size exclusion chromatography studies revealed that visfatin treatment significantly enhanced the NALP3 inflammasome complex formation and activation compared to control cells. Additionally visfatin treatment increased the caspase‐ 1 activity by 2.4 folds and IL‐1β production by 2.3 folds compared to control cells. ASC siRNA and WEHD substantially attenuated the visfatin‐induced caspase‐1 activity and IL‐1β production. In conclusion, the activation of NALP3 inflammasomes may be an important initiating mechanism responsible for the adipokine, visfatin to produce inflammatory response and endothelial dysfunction in coronary artery (supported by NIH grant HL057244, HL091464 and HL075316).
Ammonium and methyl ammonium transport by RhAG
Authors: Caner Tolga , Abdulnour-Nakhoul Solange , Brown Karen , Hamm L. Lee , Nakhoul Nazih
Published: June 2021

The erythroid Rh protein (RhAG) is one component of the “Rh complex” that is known for its immunogenic characteristics. Recently, a role of RhAG in transport of ions has been proposed. In this study, we characterized NH3/NH4+ transport by RhAG expressed in Xenopus oocytes. To do so we used ion‐selective microelectrodes & two‐electrode voltage clamp to measure cellular changes induced by NH3/NH4+ & methyl ammonium (MA/MA+). In oocytes expressing RhAG, 5mM NH4Cl (NH3/NH4+) decreased pHi by 0.28±0.04 at a rate of −9.6±0.01 ×10−4 pH/sec; depolarized the cell by 15±1.3 mV, caused an inward current of −66±8.5 nA & decreased surface pH (pHs) by 0.7±0.04. On the other hand, 5 mM MA/MA+ increased pHi by 0.15±0.02 at a rate of 12.6±2 ×10−4 pH/sec; depolarized the cell by 10.1±1.0 mV; caused an inward current of −49±11.4 nA & decreased pHs by 0.29±0.02. In H2O‐injected oocytes the changes were significantly smaller: NH3/NH4+ decreased pHi by 0.25±0.06 at a rate of only −6.0±1 ×10−4 pH/sec; depolarized the cell by 17±1.9 mV; induced an inward current of ‐ 44±5.7 nA & decreased pHs by 0.4±0.03. Notably, in H2O‐injected oocytes MA/MA+ did not elicit any change in pHi, Vm or current & decreased pHs by 0.11±0.01. These data indicate: 1) RhAG transports NH3/NH4+ & MA/MA+. 2) There is an electrogenic component of transport consistent with influx of NH4+ & MA+ by RhAG. 3) There is also evidence for electroneutral transport of NH3 & MA.
Chronic ethanol ingestion increases epithelial sodium channel activity in C57BL/6 lung
Authors: Trac David , Brewer Elizabeth , Helms My N
Published: June 2021

It is clear that a critical component of salt and water transport by the lungs depends on precise control of epithelial sodium channel (ENaC) activity, which generates the osmotic driving force for net transport of solute out of the air space. Chronic ethanol (EtOH) ingestion alters the signal transduction pathway leading to normal ENaC activity. We show that chronic EtOH ingestion in 12 week old C57BL/6 mice increases expression of ENaC, NADPH oxidase (Nox) 2, and Nox 4 protein (1.7, 1.5, and 2.8 fold, respectively) using western blot analysis. Real time PCR analysis of Rac‐guanine nucleotide exchange factors (which mediate activation of Nox) reveals that Arhgef7, Sos1, Sos2, Tiam1, and Ras‐GRF2 transcripts are significantly down‐regulated in EtOH lung compared to normal healthy lung. In vivo radiographic imaging indicates that these changes in cellular signaling lead to an enhanced rate of lung fluid clearance in EtOH animals following a tracheal saline challenge of 5uL/g body weight. We conclude that chronic ethanol ingestion activates NADPH oxidase, which oxidizes the alveolar environment in order to stimulate redox sensitive ENaC activity. This research was supported by SIRE awarded to DT, and R00‐HL‐09222601.
Prostaglandin receptor EP4 induces transient membrane targeting of aquaporin‐2 through a novel intracellular signaling pathway
Authors: Olesen Emma Tina Bisgaard , Fenton Robert Andrew
Published: June 2021

In our previous studies, the prostaglandin receptor EP2 agonist butaprost increased aquaporin‐2 (AQP2) membrane targeting (MT) and phosphorylation at ser‐269 (pS269), but EP4 agonist CAY10580 (CA) increased only MT. Potential EP4 signaling pathways alongside EP2 vs EP4 mediated AQP2 trafficking events were examined in these studies. In AQP2‐MDCK cells natively expressing EP2 and EP4, the EP2 antagonist AH6809 (10μM) abolished prostaglandin E2 (1–10nM) induced cAMP and pS269, but not MT. Stimulation with 1μM CA (2 to 80 min) increased MT from 5–80 min, but did not increase pS269‐AQP2/biotin‐AQP2 at any time point. Pretreatment with the Gi inhibitor, pertussis toxin (18h, 100ng/ml), neither increased pS269 nor affected AQP2 MT during CA stimulation. 40 min agonist washout abolished CA stimulated AQP2 MT whereas butaprost (1μM) or forskolin (10μM) induced MT was upheld for at least 80 min of washout, although pS269 was strongly decreased after 20 min. In conclusion: 1) EP4 increases AQP2 MT without increasing cAMP and pS269. This is neither due to agonist functional selectivity, concomitant Gi protein signaling nor time dependent receptor desensitization 2) EP4 mediated membrane targeted AQP2 is rapidly internalized after washout, whereas the effect of forskolin or EP2 on AQP2 MT are upheld 3) the latter is not due to continued pS269 after washout. Funded by Aarhus University and The Lundbeck Foundation.
CFTR Dependent Bicarbonate Secretion in Native Small Airway Epithelia
Authors: Shamsuddin A K M , Quinton Paul M
Published: June 2021

Loss of bicarbonate (HCO3−) transport may play a major role in pathological changes in airways, especially in Cystic Fibrosis (CF). In the small intestine normal mucus release requires CFTR dependent HCO3− secretion. HCO3− may also be crucial for effective bactericidal activities of antibiotics as well as innate antimicrobials. Evidence indicates that HCO3− secretion is present in normal, but defective in CF cultured airway cells. However, whether HCO3− is secreted in native small airways is not known. We have developed a mini‐Ussing system and assayed HCO3− transport in native small airways of pigs and humans indirectly as the changes of transepithelial potential and equivalent short circuit current. With bilateral 25 mM NaHCO3− plus 125 mM NaGlu Ringers’ with amiloride in the lumen, we stimulated with different cAMP and Ca2+ mediated agonists and inhibited with known blockers of HCO3− transport. Results showed that the native small airway epithelia are capable of secreting CFTR dependent HCO3−. HCO3− secretion is physiologically regulated, and probably critical to normal innate defense mechanism of the airways. Failure to secrete HCO3− may cause poor mucociliary and pathogen clearance and may be a target for therapy.

Supported by the Nancy Olmsted Trust, NIH (R01‐HL084042) and Cystic Fibrosis Foundation.
Biosynthetic sorting of the sodium pump: Visualization of the segregation of newly synthesized epithelial Na,K‐ATPase from apically directed proteins
Authors: Stoops Emily , Farr Glen , Caplan Michael J
Published: June 2021

Regulation of transport protein function involves many mechanisms, including endocytosis, recycling and biosynthetic sorting. The Na,K‐ATPase generates the ion gradients that drive most transepithelial transport. In almost every polarized epithelial cell type, the Na,K‐ATPase is restricted to the basolateral domain of the plasma membrane. To study the regulation of Na,K‐ATPase trafficking and its separation from apical cargo, we have created a stable MDCK cell line expressing a SNAP‐tagged version of Na,K‐ATPase and a CLIP‐tagged version of gp135, a glycoprotein that traffics to the apical membrane. The SNAP and CLIP tags are modified forms of a DNA repair protein that react with benzylguanine (SNAP) and benzylcytosine (CLIP) substrates, resulting in covalent linkage of the benzyl group to an active thiol residue within the tag. Using a block/pulse method and SNAP/CLIP‐specific blocking reagents, newly synthesized cohorts of SNAP‐ and CLIP‐tagged protein can be visualized by confocal and electron microscopy, or subjected to biochemical analysis to reveal time‐dependent protein‐protein interactions. Our data suggest that Na,K‐ATPase and gp135 pursue the same post‐synthetic trafficking pathway through the trans‐Golgi network, after which they appear to segregate from one another. Kinetic analysis reveals that Na,K‐ATPase traffics through the biosynthetic pathway more rapidly than the apical gp135.
Anaerobic Energy Stores in Emperor Penguin Muscle: Implications for Muscle Metabolism and Dive Performance
Authors: Williams Cassondra L. , Sato Katsufumi , Shiomi Kozue , Ponganis Paul J.
Published: June 2021

In diving birds and mammals, bradycardia and peripheral vasoconstriction potentially isolate muscle from the circulation. During complete ischemia, ATP production is dependent on the size of the myoglobin oxygen (O2) store and the concentrations of phosphocreatine (PCr) and glycogen (Gly). Therefore, we measured PCr and Gly concentrations in the locomotory muscle of emperor penguin and modeled the depletion of muscle O2 and those energy stores under conditions of complete ischemia, using a previously determined muscle metabolic rate. PCr and Gly concentrations were similar to published values for non‐diving animals. The model demonstrated that PCr and Gly provide a large anaerobic energy store, even for dives beyond 20 min. Although O2 consumption and energy store depletion may vary during dives, the model established that PCr and Gly are a significant anaerobic energy store and can play a vital role in the emperor penguin's ability to perform extraordinarily long dives. This work was supported by NSF grants OPP 0229638, 0538594, 0944220 and a grant from the Japanese Society for Promotion of Science (A19255001). C. Williams was supported by an NIH Marine Biotechnology Training Program Fellowship and a UC Regents Fellowship.
Histological investigations of the “slip” in marine mammal tracheas
Authors: Moore Colby Daniels , Fahlman Andreas , Moore Michael , Niemeyer Misty , Lentell Betty , Oakes Sean , Trumble Stephen
Published: June 2021

In 1940 Scholander proposed that marine mammals have stiffened upper airways that would stay open and receive air from highly compressible alveoli during diving. However, this idealized alveoli–up system would tend to underestimate the importance of tracheal collapse or the role of the trachea at pressure. There are little data available on the structural and functional adaptations of the marine mammal respiratory system. Although biomechanical and pressure‐associated changes have been measured, the aim of this research is to investigate the microscopic tracheal characteristics of five different species of diving mammals, specifically focusing on elastic fibers and distances of cartilage overlap. Our histological measurements have revealed the presence of “slip features” in both pinniped and cetacean tracheas. These “slip features” are characterized by partial or incomplete cartilaginous rings, which, at pressure, will overlap maintaining constricted airways. This finding lends evidence for pressure‐induced collapse and re‐inflation of the trachea. In some cases, more than one “slip feature” is present per tracheal ring, perhaps allowing for more rapid and effective compression. There is great variation in the anatomy of the “slip feature” between cetacean and pinniped, as some truly overlay one another and others are separated by elastic fibers, connective tissue and smooth muscle. There also seems to be a significant difference between the quantity of elastic fiber in pinniped species versus cetacean species, for example cetaceans tended to have more elastic fibers, perhaps correlating to deeper dive depths or higher ventilatory rates.
High protein diet worsens mitochondrial function and increases susceptibility to ROS‐induced permeability transition in pathological cardiac hypertrophy
Authors: Ribeiro Rogerio F , Dabkowski Erinne Rose , Galvao Tatiana F , O'Connell Kelly , Stefanon Ivanita , Stanley William C
Published: June 2021

The goal of this study was to determine the effects of high protein intake on cardiac function, mitochondrial respiration and H2O2‐induced mitochondrial permeability transition (MPTP) in pressure overload induced heart failure. Rats underwent either transverse aortic constriction (TAC) to induce hypertrophy and heart failure, or sham surgery, and were fed a diet with either 18% or 30% of energy as protein from casein. After 14 weeks ejection fraction was lower and LV mass increased in TAC vs. Sham, with no effect of diet. Isolated mitochondria showed no effect of TAC on respiration with the 18% protein diet, however the 30% protein diet decreased state III in subsarcolemmal mitochondria (SSM) with palmitoyl carnitine (sham: 202 ± 11, TAC: 185 ± 8.1, sham protein: 209 ± 9.7 and TAC protein: 152 ± 11* p<0.05). H2O2‐induced MPTP was increased by TAC in both SSM and interfibrillar mtiochondria, and significantly worsened in SSM by the 30% protein diet in TAC animals. These results suggest that high protein intake worsens mitochondrial function in pathological hypertrophy.
Metformin severely impairs in vivo muscle oxidative capacity in a rat model of type 2 diabetes
Authors: Prompers Jeanine J , Wessels Bart , Ciapaite Jolita , Nicolay Klaas
Published: June 2021

Objective

To investigate the effects of metformin on in vivo and in vitro skeletal muscle mitochondrial function in Zucker diabetic fatty (ZDF) rats using 31P magnetic resonance spectroscopy (MRS) and high‐resolution respirometry (HRR), respectively.

Methods

12‐week old healthy (fa/+) and diabetic (fa/fa) ZDF rats were treated with metformin (0, 30, 100 or 300 mg/kg body weight/day) for 15 days by oral gavage. At day 14, in vivo 31P MRS was performed on the tibialis anterior (TA) muscle to measure PCr recovery. At day 15, animals were killed and TA muscles were excised for in vitro HRR measurements.

Results

Metformin treatment decreased PCr recovery rates in a dose‐dependent manner in both healthy fa/+ and diabetic fa/fa rats. Whereas, the clinical dose of 30 mg/kg/day had no significant effect, PCr recovery rates were ~22% and ~47% decreased at 100 and 300 mg/kg/day. HRR measurements showed a similar, but less pronounced effect of metformin on in vitro mitochondrial function.

Conclusion

Metformin treatment impairs in vivo skeletal muscle oxidative capacity in rats in a dose‐dependent manner. This research was funded by a VIDI grant from the Netherlands Organisation for Scientific Research (NWO).
Mitochondrial Ogg1 prevents loss of mitDNA content, decreases mitochondria fission and apoptosis under conditions of oxidative stress in H9C2 cells
Authors: Torres-Gonzalez Moises , Gawlowski Thomas , Scott Brian , Dillmann Wolfgang
Published: June 2021

Oxidative stress induces damage and depletion of mitochondrial (mit) DNA and suppresses ATP production leading to defects in cell function and initiating cell death. 8‐oxoguanosine DNA glycosylase‐1 (OGG1) acts in the mitochondria to repair DNA damage. In this study H9C2 cells were infected with either adv‐ OGG1 or empty vector and, challenged with menadione, to induce mitochondrial DNA damage. The cells were used to evaluate the effects of OGG1 on mitochondrial DNA content and damage and on apoptotic events induced by oxidative stress. Levels of 8‐oxodG and AP‐sites, metrics of mitochondrial DNA damage, were decreased by 30% and 35%, respectively, in adv‐Ogg1 infected cells. Cells over‐expressing mitOgg1 showed increased membrane potential (p<0.05) and decreased mitochondrial fragmentation (p<0.005). mitOgg1 over‐expression preserved mitDNA content and lowered levels of fission and apoptotic factors such as DRP‐1, FIS1, cytoplasmic cytochrome c, caspase‐3 and caspase‐9. These observations suggest that mitOgg1 may be an alternative to protect cardiac cells against oxidative stress damage.
Isoflurane Increases Mitochondrial Free Ca2+ by Attenuating the Na+/Ca2+ Exchanger Activity
Authors: Agarwal Bhawana , Camara Amadou K.S. , Stowe David F. , Bosnjak Zeljko J. , Beard Daniel A. , Dash Ranjan K.
Published: June 2021

Volatile anesthetics protect against cardiac ischemia/perfusion (IR) injury. Excess mitochondrial free Ca2+ ([Ca2+]m) is a major cause of mitochondria ‐mediated IR injury. To better understand mechanisms of anesthetic ‐mediated protection of mitochondrial function, we monitored time courses of NADH redox state, respiration, ΔΨm, and matrix Ca2+ uptake/efflux kinetics during anesthetic exposure. Isolated mitochondria from rat hearts were energized with 10 mM Na+‐or K+‐pyruvate+malate, or with Na+‐succinate followed by isoflurane (0.5 to 2 mM), CaCl2 (≈ 200 nM free Ca2+), and ADP (250 μM). Our data showed that isoflurane: (a) increased [Ca2+]m despite ΔΨm depolarization, while decreasing Na+‐dependent Ca2+ efflux (NCE), (b) decreased state 3 NADH oxidation and increased state 3 duration with Na+‐pyruvate/malate, but c) caused no change in state 3 NADH oxidation but reduced state 3 duration with Na+‐succinate. These data indicate that isoflurane (1) increases [Ca2+]m by attenuating activity of NCE and, (2) decreases the rate of ADP phosphorylation while reducing the duration of state 3 respiration by attenuating complex I activity. Isoflurane's effects on inhibiting NCE and complex I together may act as the protective mechanism against IR injury at the mitochondrial level.
(Pro)renin receptor over‐expression induces angiotensin II‐independent NADPH oxidase activation through PI3K ‐ ERK signaling in Neuronal cells
Authors: Peng Hua , Li Wenchen , Seth Dale , Gabriel Navar L , Feng Yumei
Published: June 2021

We previously showed that (pro)renin receptor (PRR) mediates angiotensin II‐independent NADPH oxidase (NOX) activation and oxidative stress in neuronal cells. To investigate the signaling cascade of these events, Neuro‐2A cells were infected with adeno‐associated virus coding for human PRR and control virus. Cells were then treated with phosphoinositide 3‐kinase (PI3K,) inhibitor (wortmannin, 2uM) or extracellular signal‐regulated kinases 1/2(ERK1/2) inhibitor (U0126, 20uM). NOX activity, NOX2 and NOX4 expression levels were determined. The PI3K and ERK1/2 inhibitor decreased NOX2 (wortmannin: 2.685±0.11 vs. 6.346±0.532, P<0.05; U0126:0.373±0.03 vs. 6.346±0.53, P<0.05) and NOX4 (wortmannin:1.803±0.04 vs. 2.658±0.13, P<0.05; U0126:1.391±0.01 vs. 2.658±0.13, P<0.05) mRNA levels (Relative fold change) compare to vehicle following PRR over‐expression. Furthermore, PI3K (0.965±0.02 vs. 1.280±0.03, P<0.05) or ERK1/2 (0.923±0.03 vs. 1.280±0.03, P<0.05) inhibition significantly reduced NOX activity (Relative luminescence units) compared to vehicle following PRR over‐expression. These data suggested that PRR over‐expression induces oxidative stress in neuronal cells through activation of PI3K ‐ERK1/2 signaling cascade, and thus up‐regulation of NOX2 and NOX4 levels, indicating a new pathway for oxidative stress in the central nervous system.(NIH1P30HL101285, AHA11SDG73600050)
Downregulation of AT1 receptor in the RVLM induced by mesenchymal stem cells treatment prevent the hypertension and sympathetic hyperactivity in 2K‐1C Wistar rats
Authors: Oliveira-Sales Elizabeth Barbosa , Maquigussa Edgar , Semedo Patricia , Oliveira Luciana G. , Câmara Niels O.S. , Bergamaschi Cassia T. , Boim Mirian A , Campos Ruy R
Published: June 2021

Current approaches to pharmacological treatment of renovascular hypertension are problematic and thus, alternative therapies are needed. We investigated the effects of mesenchymal stem cells (MSC) treatment on systolic arterial pressure (SAP), renal sympathetic nervous activity (rSNA) and AT1 receptor (AT1R) expression in the rostral ventrolateral medulla (RVLM) in an experimental model of renovascular hypertension, 2 Kidney‐1 clip (2K‐1C). Fluorescently tagged MSC (2×105cells/animal, IV) were injected weekly after the 3rd week of renal artery occlusion and the experiments were performed at the end of 6 weeks after clipping. The rats were divided in control group (n=5), control group treated with MSC (n=2), 2K‐1C (n=8) and 2K‐1C treated (n=7). Bilateral punches were taken from the RVLM to perform AT1R protein expression analyses by western blotting. Tracking assays by citometry showed MSC in the medulla oblongata. SAP in 2K‐1C‐treated rats was lower compared to untreated animals (2K‐1C, 224 ± 8 vs 173 ± 6 mmHg, p<0,05) at 6th week followed by a significant decrease in rSNA (27%). MSC treatment reduced brainstem AT1R expression only 2K‐1C rats (28%, p<0,05). Therefore, the results suggest MCS treatment can both prevent the increase in SAP as well as reduce the sympathetic hyperactivity and the expression of AT1R in the RVLM, constituting a future alternative for the treatment of this type of hypertension.

Supported by CAPES