bluuebunny/crossref_metadata_2025 huggingface dataset:
AMPK activation by a novel small molecule of mitochondrial regulation enhances fatty acid oxidation, ketogenesis and branched chain amino acid catabolism in vivo
Authors: Hitoshi Yasumichi , Jenkins Yonchu , Markovtsov Vadim , Sun Tian‐Qiang , Li Yingwu , Pan Alison , Uy Gerald , Gross Lisa , Li Wei Q , Baltgalvis Kristen , Nguyen Henry , Gururaja Tarikere , Kinoshita Taisei , McCaughey Kelly , White Kathy , Godinez Guillermo , Alcantara Raniel , Owyang Alexander , Choy Carmen , Boralsky Luke , Goff Dane A , Shaw Simon , Singh Rajinder , Kinsella Todd , Payan Donald G
Published: June 2021

The 5′‐AMP‐activated protein kinase (AMPK) is a key regulator of energy homeostasis. Pharmacologic activation of AMPK is beneficial in treating metabolic disorders and improving exercise endurance. We previously identified small molecule AMPK activators that improved glucose tolerance and running endurance. These compounds reduced cellular oxygen consumption and increased AMP/ATP and ADP/ATP ratios, demonstrating that AMPK activation is due to a change in energy status which results from altered mitochondrial respiration. Further biochemical studies showed that the target appeared to be mitochondrial complex I. In vivo metabolite profiling indicated that there was an up‐regulation of fatty acid oxidation, ketogenesis and catabolism of branched chain amino acids in the compound‐treated mice. The up‐regulation in fatty acid catabolism was supported by a clear reduction of fat pads in treated mice. Furthermore, UCP1 protein expression was increased in the white adipose tissues. Taken together, our results demonstrate beneficial outcomes by AMPK activation through mitochondrial regulation. These studies should help to provide a molecular understanding for the physiological effects of AMPK activation and for the therapeutic applications of AMPK activators.
StaR engineering: GPCR stabilisation for structure‐based drug design
Authors: Wiggin Giselle , Patel Jayesh C , Kean James , Khan Samir Ali , Mould Richard , Brown Jason , Ng Irene W , Errey James C , Dore Andrew S , Zhukov Andrei , Tehan Benjamin , Christopher John A , Jazayeri Ali , Marshall Fiona H
Published: June 2021

G‐protein‐coupled receptors (GPCRs) represent one of the largest and most important classes of membrane proteins for drug discovery. However, due to their inherent flexibility and instability outside of the membrane, they are challenging targets for structural and biophysical studies. Here we demonstrate how these challenges have been overcome through protein engineering, by identifying a minimal number of thermostabilising mutations that can lock a receptor in a single conformation and enhance its survival in a detergent environment. This has been applied to GPCRs from families A, B and C, with peptide or small molecule ligands, and to orphan receptors with no known ligand. The resulting stabilised receptors (StaRs) are more amenable to purification, crystallisation and biophysical analysis of ligand binding.

Using this approach, StaRs were generated for Orexin 1 and Orexin 2, two peptide GPCRs involved in regulating the sleep‐wake cycle. The StaRs show decreased agonist signalling, whilst maintaining antagonist binding, consistent with a shift to the antagonist conformation. Structural determination, together with biophysical mapping and fragment screening using surface plasmon resonance (SPR), has led to the rapid identification of highly optimised dual Orexin receptor antagonists (DORAs) with very high affinity, good pharmacokinetics and excellent in vivo activity in models of insomnia.
Different classes of CB1 ligands bias CB1‐dependent signal transduction
Authors: Laprairie Robert Brad , Dupre Denis J , Kelly Melanie EM , Denovan‐Wright Eileen M
Published: June 2021

Biased agonism describes the ligand‐dependent selectivity of different signal transduction pathways by molecules that act as agonists at the same receptor. This functional selectivity may be exploited to alter the balance between signaling pathways downstream of a receptor. Agonists of the type 1 cannabinoid receptor (CB1) are structurally diverse and include the aminoalkylindole WIN55,212–2, the endocannabinoid N‐arachidonoyl ethanolamine (anadamide), and the Δ9‐tetrahydrocannabinol (THC)‐like, CP55,940. Given the differences in potency and efficacy of these agonists, we sought to determine whether activation of CB1 by these ligands resulted in biased signal transduction in a cell culture model of striatal neurons. We found that the THC‐like compound CP55,940 enhanced the interaction between CB1 and β‐arrestin2 compared to vehicle, anandamide, and WIN55,212–2, which resulted in CB1 internalization and persistent pERK signalling. In contrast, the endocannaibnoid anandamide and the aminoalkylindole WIN55,212–2 enhanced the interaction between CB1 and Gαi, and promoted a rapid and transient increase in pERK signaling compared to vehicle or CP55,940. Based on these data, the therapeutic efficacy of cannabinoid agonists may be maximized by selecting agents with specific potency, affinity, and signaling bias.
R‐ and S‐ mTFD‐MPPB, an Anesthetic and a Convulsant, Have Opposing Effect on γ‐Aminobutyric acid (GABA) type A Receptors
Authors: Desai Rooma , Savechenkov Pavel , Ge Rile , Bruzik Karol , Raines Douglas , Miller Keith
Published: June 2021

The R‐enantiomer of 1‐methyl‐5‐phenyl‐5‐propyl barbituric acid (MPPB) is an anesthetic whereas the S is a convulsant. We synthesized its trifluoromethyl derivative (mTFD‐MPPB), a novel photoactivable barbiturate. R‐ and S‐mTFD‐MPPB caused loss of righting reflex in tadpoles (EC50 28 μM & 40 μM respectively) but the S‐enantiomer caused excitability. Next we used whole‐cell patch clamp electrophysiology with fast solution exchange (<1 millisecond) on recombinant human α1β3γ2 GABAA receptors expressed in HEK cells and found both enantiomers acted on GABAA currents more potently than MPPB. Currents elicited by low GABA concentration (10 μM) were increased 5‐fold by RmTFD‐MPPB (46μM), but decreased by ~70% by S‐mTFD‐MPPB (46μM). Enhancement was never observed with S‐mTFD‐MPPB at any concentration tested. At high GABA concentration (10 mM), both mTFD‐MPPB enantiomers (46μM) inhibited currents by ~30%. The enantiomers’ actions at low GABA are more consistent with their pharmacology, suggesting that the physiological locus of action is a GABAAR that functions at low GABA occupancy or where GABA is a partial agonist. This might include extrasynaptic receptors such as those containing δ‐subunits. Supported by GM58448.
Targeting Inhibitor of Protein Phosphatase 2A (I2PP2A) Mediates Plasma Membrane Beta‐Adrenergic Receptor Resensitization
Authors: Martelli Elizabeth Eve , Vasudevan Neelakantan T , Mohan Maradumane L , Gupta Manveen K , Naga Prasad Sathyamangla V
Published: June 2021

βAR downregulation and desensitization are hallmarks of heart failure. Agonist occupied βARs undergo desensitization through phosphorylation by G‐protein couples receptor kinases leading to βAR internalization. Phosphorylated βAR becomes resensitized following dephosphorylation by PP2A in the endosome and is the paradigm in receptor resensitization. Our recent studies have suggested that resensitization of βARs can occur on the plasma membrane through the inhibition of PP2A by I2PP2A. We generated a stable HEK (Human Embryonic Kidney) cell line expressing short hairpin RNA targeting I2PP2A (shRNA‐I2PP2A). Confocal microscopy studies show that cells expressing shRNA‐I2PP2A resulted in significant loss of receptor phosphorylation despite the presence of the agonist. Furthermore, radioligand binding and confocal imaging showed marked inhibition of receptor internalization upon depletion of I2PP2A that was associated significantly with PP2A activation. Consistently, we also observed preservation of βAR function despite the presence of agonist as measured by cAMP generation and adenylyl cyclase activity. Together, these studies suggest that targeting I2PP2A may lead to plasma membrane receptor resensitization demonstrating that modulating PP2A function can potentially change desensitization and resensitization dynamics of the receptors on the plasma membrane. NIH/NHLBI: 1 RO1 HL 08943, 1 RO1 HL 115008
Role of Gravin Scaffolding of PKC Signaling in the Heart
Authors: Singh Sonal , Guillory Ashley , Wijaya Cori , McConnell Bradley K.
Published: June 2021

Gravin, an A kinase anchoring protein, scaffolds p r o t e i n k i n a s e A ( PKA), protein kinase C (PKC), and other signaling molecules to the β2‐adrenergic receptor (AR). Agonist stimulation of β2‐AR leads to gravin‐dependent desensitization of the receptor. Disruption of PKA/AKAP interaction or suppression of gravin expression disrupts β2‐AR desensitization by PKC phosphorylation. The objective of this study was to investigate the role of PKC on cardiac function in the absence of gravin mediated scaffolding. Total PKC activity was determined in wild‐type and gravin knockout mice stimulated with isoproterenol (30mg/kg/day) for 14‐days. Basal PKC activity was significantly increased in the KO animals. Additionally, PKC activity significantly increased in both WT and KO ISO groups compared to their respective controls. PKC activity was also found to be greater in the KO ISO group as compared to both the KO and the WT controls (p<0.0001). However, despite of the changes in PKC activity, no change in the expression of PKCα was seen in the KO mice, either before or after ISO treatment. Hence in the absence of scaffolding mediated by gravin, there is an increase in the PKC activity without a corresponding change in PKCα expression or phosphorylation. Further investigation is being carried out using PKC activators and inhibitors to determine the interplay between gravin and PKC in modulating the PKC signaling in heart.

Source of research support was RO1HL085487.
Therapeutic potential of KIOM‐62 extract for atopic dermatitis in vitro and in vivo
Authors: Lim Hye‐Sun , Ha Hyekyung , Lee Mee‐Young , Jeong Soo‐Jin , Jin Seong Eun , Shin* Hyeun‐Kyoo
Published: June 2021

KIOM‐62 (Compositae) is a traditional herbal medicine used for treatment of inflammatory diseases such as rheumatoid arthritis, chronic gastritis, and asthma. However, their topical effect on atopic dermatitis has not been studied yet. The aim of this study was to investigate the effects of KIOM‐62 on atopic dermatitis using HaCaT human keratinocyte cells and NC/Nga mice model of house dust mites‐induced atopic dermatitis. We found that KIOM‐62 suppressed the overexpressed of macrophage‐derived chemokine (MDC/CCL22) protein as well as mRNA induced by TNF‐α and IFN‐γ in HaCaT cell. In addition, atopic dermatitis was induced by topical application of house dust mite extract on ear and back skin of NC/Nga mice twice a week for 6 weeks. KIOM‐62 was administered daily by oral gavage for 6 weeks with induction of atopic dermatitis. KIOM‐62‐treated mice showed the reduction in plasma IgE level and the severity of dermatitis (erythema/hemorrhage, edema, erosion, and scaling) compared with atopic dermatitis‐induced group. KIOM‐62‐treated mice also attenuated atopic dermatitis‐like skin lesions such as erosion, hyperplasia of the epidermis and dermis, and inflammatory cell infiltration on the skin of back and ear in histological examination. Taken together, these results suggest that KIOM‐62 may be a potential therapeutic modality for atopic dermatitis.
Discovery of a novel enhydrazone ester (CEE‐1) that acts as a dual inhibitor of the release of pro‐inflammatory cytokines and prostanoids from human monocytes
Authors: Ezeamuzie Charles , Zamil Hanan , Al‐Baghli Hawraa , Edafiogho Ivan O
Published: June 2021

CEE‐1 (ethyl 4‐phenylhydrazinocyclohex‐3‐en‐2‐oxo‐6‐phenyl‐1‐oate) ‐ a novel enhydrazone ester, was synthesized and tested in vitro for anti‐inflammatory activity against the release of pro‐inflammatory cytokine and prostanoid from activated human monocytes. The effects were compared with those of standard anti‐inflammatory drugs dexamethasone and indomethacin. CEE‐1 potently and strongly inhibited the release of both TNF‐α and PGE2. The IC50 values were 2.6 μM and 4.5 μM for TNF‐α and PGE2, respectively. At 30 μM, the drug achieved almost complete inhibition of both mediators. Dexamethasone had similar effects but indomethacin inhibited only the PGE2 release, and although CEE‐1 was less potent than these two drugs, it had comparable efficacy. The compound appeared to act, at least, in part by inhibiting the up‐regulation of the mRNA for TNF‐α as well as that of the prostanoid‐synthetic enzyme, COX‐2. However, like dexamethasone, but unlike indomethacin, CEE‐1 did not affect COX‐2 enzyme function. Thus, CEE‐1 acts more like steroids rather than the non‐steroidal anti‐inflammatory drugs. Structure‐activity study showed that the presence of a simple aromatic ring attached via an NH‐NH group was critical for activity. At the concentrations that completely inhibited mediator release, the compound displayed no significant in vitro toxicity on the cells. These results show that CEE‐1 is a dual inhibitor of the release of cytokines and prostanoids, and therefore could be a potential alternative to steroids in the treatment of inflammatory diseases.

This work was supported by Kuwait University, Research Sector grant # MR 03/09.
Direct Electrochemistry of Shewanella oneidensis Cytochrome c Nitrite Reductase
Authors: Judd Evan Thomas , Elliott Sean J
Published: June 2021

Shewanella oneidensis cytochrome c nitrite reductase (soNrfA) is a dimeric enzyme that plays a key role in bacterial nitrate respiration. The periplasmic reductase catalyzes the six‐electron reduction of nitrite to ammonia but can also catalyze the two‐electron reduction of hydroxylamine, a proposed intermediate in the nitrite reduction pathway. Here we use Protein Film Voltammetry to investigate the electrochemical and catalytic properties of soNrfA and compare its properties to the related Escherichia coli nitrite reductase (ecNrfA). Our findings highlight the similarities and differences between the two enzymes: most notably, all detected electron transfer steps are one‐electron in nature, contrary to what has been observed in ecNrfA. This suggests that for soNrfA the six‐electron reduction of nitrite is carried out in a series of one‐electron steps. Additionally we observe substrate inhibition during nitrite turnover and evidence of negative cooperativity during hydroxylamine turnover which suggests individual monomers within a ccNiR homodimer interact with one another during catalysis.
Pharmacological evidences of the involvement of calcium channels in (−)‐β‐Pinene‐induced vasodilatation
Authors: Santos Marcio Roberto , Moreira Italo Jose , Cunha Patricia Santos , Santos Sandra Lauton , De Lucca Waldecy , Ribeiro Eurica Adelia , Santana‐Filho Valter Joviniano , Passos‐Junior Daniel Badaue , Araujo Adriano Antunes , Quintans‐Júnior Lucindo Jose
Published: June 2021

(−)‐β‐Pinene is a monoterpene found in various herbs with activity on the cardiovascular system. Pharmacological proprieties such as miorelaxant and hypotensive were related. This study aimed to evaluate the vasodilatation effect of the (−)‐β‐pinene in rings of rat mesenteric artery. Wistar rats were euthanized and superior mesenteric artery was removed and cut in rings (1–2 mm). These rings were then mounted in organ baths containing Tyrode's solution at 37 ºC and gassed with carbogen. For isometric tension recordings, each ring was fixed in a force transducer connected to an acquisition system. All procedures described in this work were approved by the Ethical Committee on Animal Experiments from Federal University of Sergipe, Brazil (n° 72/10). In rings pre contracted with phenylephrine, (−)‐β‐pinene (10−7 – 10−2 M) was able to induce relaxations (Emax = 104 ± 16%; n = 6) that was not affected after removal of the endothelium (Emax = 108 ± 4%; n = 6), in rings without endothelium pre‐contacted with KCl 80 mM (Emax= 112 ± 14%; n = 6), or after incubation with tetraethylamonium (Emax= 113 ± 3%; n = 6). Furthermore, (−)‐β‐pinene was able significantly (p < 0.05) to inhibit the CaCl2‐ or Bayk‐8644‐induced contractions. In conclusion, these results demonstrate that (−)‐β‐pinene induces vasodilatation possibly caused by the inhibition of the Ca2+ influx through voltageoperated Ca2+ channels.
Primary Human Cardiomyocyte Culture: A model for assessment of cardiovascular disorders
Authors: Collins Atijah , Scott Jessica , Gunturi Rahul , Sharma Avadhesh C.
Published: June 2021

The purpose of this research is to establish an in vitro cardiac system using primary human cardiomyocyte cultures (PHCC). We hypothesized that norepinephrine (NE) and endothelin‐1 (ET‐1) will contribute to human cardiomyocyte growth. PHCC were obtained from Celprogen, San Pedro, CA and grown with varied concentrations of NE or ET‐1 for 72h. Protein expressions of contractile proteins were analyzed via Immunoblot and immunocytochemistry. Analysis of confocal images showed a significant decrease in nuclear area in all NE treatment groups. NE (10 μmol) stimulated significant increases in fluorescent intensities of the contractile proteins, f‐actin, troponin and tropomyosin. The protein expression of myosin light chain was significantly lower with increasing concentrations of NE. In addition, the average cell area increased in all groups of ET‐1. ET‐1 (1, 10, 100 nmol) exhibited a significant decrease of fluorescent intensities of f‐actin, troponin and tropomyosin. PHCC were also grown in the presence of LPS (1, 10, 100μg/ml) in a 6‐well plate for 72h. Viability data showed the cells recovered from the endotoxin treatment and maintain steady growth in each concentration of LPS. In contrast to NE, ET‐1 exhibited a decrease on the expression of contractile proteins in PHCC.
Curcumin Induces Autophagy to Protect Vascular Endothelial Cell Survival from Oxidative Stress Damage
Authors: Li Xue‐Jun , Han Jing
Published: June 2021

Our study first proposed that curcumin could protect human endothelial cells from the damage caused by oxidative stress via autophagy. Furthermore, our results revealed that curcumin causes some novel cellular mechanisms that promote autophagy as a protective effect. Pretreatment with curcumin remarkably improves the survival of human umbilical vein endothelial cells (HUVECs) from H2O2‐induced viability loss. Exposed to H2O2, curcumin‐treated HUVECs upregulate the level of microtubule‐associated protein 1 light chain 3‐II (LC3‐II), the number of autophagosomes, and the degradation of p62. Curcumin can also reverse FOXO1 (a mediator of autophagy) nuclear localization along with causing an elevated level of cytoplasmic acetylation of FOXO1 and the interaction of acetylated FOXO1 and ATG7, under the circumstance of oxidative stress. Additionally, knockdown of FOXO1 by shRNA inhibits not only the protective effects that curcumin induced, but the autophagic process, from the quantity of LC3‐II to the expression of RAB7. These data uncover a brand new protective mechanism involving FOXO1 as having a critical role in regulating autophagy in HUVECs, and suggest a novel role for curcumin in inducing a beneficial form of autophagy in HUVECs, which may be a potential multitargeted therapeutic avenue for the treatment of oxidative stress‐related cardiovascular diseases. This work was supported by the National Natural Science Foundation of China.
Transcriptional regulation of cardiac cell death and survival signaling by helium postconditioning in a rat model of regional cardiac ischemia/reperfusion
Authors: Oei Gezina T.M.L. , Heger Michal , van Golen Rowan F. , Preckel Benedikt , Hollmann Markus W. , Weber Nina C.
Published: June 2021

Helium is a non‐anesthetic, noble gas, which has long been used in humans without deleterious side effects. In animal models of regional myocardial ischemia/reperfusion (I/R), helium inhalation during early reperfusion (postconditioning) reduces infarct size. To gain mechanistic insight into this process we investigated the expression of genes involved in cell death and survival pathways after helium postconditioning.

Male rats (n=8 per group) were subjected to ischemia, I/R, or I/R and 15 min of 70% helium at the onset of reperfusion. The sham group (Sham) did not undergo I/R. Gene expression profiles related to necrosis, apoptosis and autophagy were assayed with qRT‐PCR using PCR arrays. Data were expressed as fold increase or decrease versus the I/R group.

Helium postconditioning caused upregulation of genes involved in necrosis (17 of 23) and pro‐apoptosis (18 of 25). Simultaneously, 4 of 23 (necrosis) and 7 of 25 genes (pro‐apoptosis) were downregulated. The majority of anti‐apoptotic genes (9 of 11) and genes involved in autophagy (24 of 32) was upregulated after helium postconditioning.

These data suggest that helium postconditioning induces cell survival pathways to such an extent that execution of cell death programs is partially abrogated or offset, leading to a reduction in infarct size.
Electron Distribution within the Cytochrome c Nitrite Reductase Hemes as a Function of Applied Potential: a Spectro‐Potentiometric Analysis
Authors: Stein Natalia , Bennett Brian , Pacheco Andy
Published: June 2021

Cytochrome c Nitrite Reductase (CcNiR) is a dimeric multi‐heme enzyme that catalyzes the reduction of nitrite to ammonia. The ten heme moieties within the dimer are arranged in such proximity to each other as to form a wire that is used to transfer electrons from a physiological donor to substrate bound at the unique active site. This poster will present two results from recent studies: the effect of added cyanide on the ccNiR heme midpoint potentials, and the binding affinity of the oxidized ccNiR active site for nitrite and cyanide. These studies form part of a larger mechanistic study of the enzyme. UV/Vis spectropotentiometric titrations of ccNiR in the presence and absence of cyanide reveal 5 distinct Nernstian potentials that can be modeled as 1‐electron reduction steps. When cyanide is bound to the active site the midpoint potential of the highest‐potential heme shifts nearly 70 mV in the positive direction, while the potentials of the next two hemes each shift by about 25 mV in the same direction. The two lowest‐potential hemes are unaffected by cyanide. Spectral analysis suggests that in all cases individual electrons are distributed among several hemes, rather than being localized on a single heme. The spectropotentiometric titrations are now being repeated using electron paramagnetic resonance (EPR) spectroscopy in place of UV/Vis, in order to obtain detailed information about individual hemes.
Protective mechanisms of atorvastatin in ameliorating doxorubicin‐induced hepato‐renal toxicity
Authors: El‐Moselhy Mohamed Ahmed Mohamed , El‐Sheikh Azza A.K.
Published: June 2021

This study aimed to investigate the mechanisms by which the anticancer drug doxorubicin (Dox)‐induced hepato‐renal damage could be prevented by Atorvastatin(Ator), a cholesterol‐lowering statin. Ator (10 mg/kg for 10 days) was administered orally, and, in independent rat groups, Dox hepato‐renal toxicity was induced via a single i.p. dose (15 mg/kg at day 5 of experiment), with or without Ator. Dox caused deterioration in hepato‐renal function, as it increased BUN, creatinine, ALT and AST levels compared to control. Dox also caused distortion in normal renal and hepatic histology, with significant oxidative stress, as it decreased GSH concentration and catalase activity and increased malondialdehyde level. In addition, Dox caused nitrosative stress, as it increased No level, with down‐regulation of eNOS. Furthermore, Dox caused inflammatory effects as shown by up‐regulation of hepato‐renal NF‐κB expression and increment of TNF‐α concentration. Dox also caused apoptotic effect, as it up‐regulated Bax expression in liver and kidney. Using Ator with Dox restored hepato‐renal functions and histological structure, also reversed oxidative/nitrosative stress markers, inflammatory signs and apoptosis. These findings suggest Ator as a protective adjuvant against Dox toxicity, via antioxidant, anti‐nitrosative, anti‐inflammatory and anti‐apoptotic mechanisms.
Effects of angiotensin II type 2 receptor agonist on stretch‐induced ANP secretion via PI 3K/PKG/NO pathway
Authors: Gao Shan , Park Bung Mun , Cha Seung Ah , Park Woo Hyun , Kim Suhn Hee
Published: June 2021

Angiotensin II (Ang II) is well‐known a major peptide of renin‐angiotensin system (RAS) as regulating blood pressure and body fluid volume. It is already known that the AT1 receptor mediates the major cardiovascular effects of Ang II. However, effects mediated via AT2 receptor are still controversial. The aim of the present study is to define the effect of AT2 receptor agonist, CGP 42112A on high stretch‐induced ANP secretion and its mechanism using in vitro and in vivo experiments. CGP 42112A stimulated high stretch‐induced ANP secretion and concentration from isolated perfused rat atria. The augmented effect of CGP42112A (0.1 μM) on high stretch‐induced ANP secretion was attenuated by the pretreatment with AT2 receptor antagonist or inhibitor for phosphoinositol 3‐kinase (PI3K), nitric oxide (NO), soluble guanylyl cyclase (sGC), or protein kinase G (PKG). However antagonist for AT1 or Mas receptor tended to augment the effect of CGP 42112A on high stretch‐induced ANP secretion. In vivo study, acute infusion of CGP 42112A for 10 min increased plasma ANP level without blood pressure change and pretreatment with losartan for 10 min followed by CGP 42112A infusion increased plasma ANP level more than CGP 42112A alone. In hypertensive atria, AT2 receptor mRNA and protein level were up‐regulated. The response of plasma ANP level by acute infusion of CGP 42112A in hypertensive rat augmented. Therefore, we suggest that AT2 receptor agonist stimulates high stretch‐induced ANP secretion through PI3K/NO/sGC/PKG pathway.

Supported by the National Research Foundation (2012–0009322)
Phosphatase 2A is involved in Adherens Junction (AJ) Regulation in Endothelial Cells
Authors: Kasa Anita , Czikora Istvan , Gergely Pal , Verin Alexander D. , Csortos Csilla
Published: June 2021

We have previously shown that Ser/Thr phosphatase 2A (PP2A) is important for endothelial cell (EC) barrier integrity preservation. To further define the functional role of PP2A in EC barrier we examined the role of PP2A in the regulation of EC AJs. EC immunofluorescence staining revealed the presence of Bα subunit in the AJs area. Moreover specific interaction between PP2AB and AJs proteins was detected by pull down assay. Inhibition of PP2A negatively affected the EC function, in concert with the decreased expression of VE‐cadherin, and β‐catenin in the AJs and inreased β‐catenin phosphorylation. The depletion of Bα (regulatory) PP2A subunit led to the translocation of AJs proteins from the cell junctions to the cytoplasm in parallel with β‐catenin phosphorylation and F‐actin remodeling, suggesting a regulatory role of PP2A in EC barrier function and cytoskeletal integrity. The effect of Bα depletion was further enhanced by thrombin treatment. Collectively, these data strongly suggest that PP2ABα has a functional role in AJs regulation via the dephosphorylation of β‐catenin. These results also support a critical role of Bα plays in the barrier‐protective function of PP2A.

MLOG7307
Effect of resveratrol and caloric restriction on mitochondrial regulation within different adipose tissues in aged rats
Authors: Malamo Angelina G , Carey‐Love Sean , Wawrzyniak Nick , Silvestre Jason , Nguyen Linda M.‐D. , Joseph Anna‐Maria , Dutta Debapriya , Xu Jinze , Leeuwenburgh Christiaan , Adhihetty Peter J.
Published: June 2021

It is well established that impairments in mitochondrial function/regulation contribute to tissue decline with aging. Caloric restriction (CR) and resveratrol (RSV) treatment in rodents induces beneficial mitochondrial adaptations in tissues such as heart and skeletal muscle but whether similar benefits occur in fat is unknown. Thus, we investigated whether RSV (50 mg/kg/day; 6 weeks) and/or CR (20% reduced AL; 6 weeks) could alter mitochondrial regulation/biogenesis in aged rodent adipose tissues (visceral; VIS, epididymal; EPI, brown adipose tissue; BAT). Aged F344xBN rats (26 mo) were divided into 4 groups (n=4): ad libitum (AL), CR, RSV, RSV+CR and mitochondrial content (cyto c, COX activity, and COX I) and mitochondrial signaling/regulation (AMPK, PGC‐1α) were assessed. Expectedly, mitochondrial content (cyto c, COX activity) and PGC‐1α were significantly elevated (~2–3‐fold) in the BAT compared to EPI and VIS fat. CR and RSV tended to increase COX activity (~20–50%) in all adipose depots but showed inconsistencies with other mitochondrial content/signaling markers. Interestingly, combined CR and RSV treatment had no effect and/or suppressed COX activity in all adipose tissues. Our data indicates that short‐term CR and RSV treatment when applied independently appears to enhance mitochondrial content in adipose tissue and may contribute to improvements in health associated with these paradigms.
Effect of chronic alcohol feeding on physiological and molecular parameters of thiamin uptake by pancreatic acinar cells of transgenic reporter mice carrying the human SLC19A2 and SLC19A3 promoters
Authors: Srinivasan Padmanabhan , Subramanian Veedamali S , Said Hamid M
Published: June 2021

Thiamin is essential for metabolic functions of pancreatic acinar cells. These cells obtain the vitamin from their surrounding via a specific carrier‐mediated process that involves THTR‐1 and THTR‐2 (products of SLC19A2 and SLC19A3 genes, respectively). We have previously shown that chronic alcohol exposure inhibit thiamin uptake by these cells but little is known if the effect is mediated at the transcriptional level of the involved transporters. We addressed this issue using transgenic mice carrying the human SLC19A2 and SLC19A3 promoters and pancreatic acinar 266–6 cells expressing these human promoters. Chronic alcohol feeding (4 weeks) of transgenic mice led to a significant inhibition in carrier‐mediated thiamin uptake, a reduction in level of the expression of endogenous mouse THTR‐1 and THTR‐2 at the protein and mRNA levels, and a significant reduction in activity of SLC19A2 and SLC19A3 promoters. Similar findings were observed in alcohol exposed 266–6 cells. In the latter model, we were also able to determine the alcohol sensitive region within the SLC19A2 and SLC19A3 promoters using a series of truncated promoter constructs. These studies provide clear evidence that the effect of chronic alcohol exposure on pancreatic acinar thiamin uptake is mediated at the level of transcription of SLC19A2 and SLC19A3 genes. [Supported by DVA and NIH grants DK56061 and AA018071].
Environmental enrichment modulates hormonal and behavioral responses induced by chronic stress in rats
Authors: Costa Rafaela , Cunha Tatiana Sousa , Casarini Dulce Elena , Marcondes Fernanda Klein
Published: June 2021

The aim of this study was to evaluate the effect of 2h daily exposition to environmental enrichment (EE) during 5 weeks on hormonal and behavioral responses induced by chronic mild and unpredictable stress (CMS) applied during 3 consecutive weeks in rats. Male adult rats were divided into 4 groups: control (C), EE, CMS and CMS + EE. Depression‐like behavior was evaluated in the forced swimming test (FST) and memory was evaluated by Y‐maze test at the end of protocol. One day after behavioral tests, the animals were killed and trunk blood was collected for corticosterone assay. All procedures were approved by Institutional Ethics Committee. Two‐way ANOVA + Tukey test was used, p<0.05. CMS (50.8 ± 6.1) and CMS + EE (18.0 ± 5.1) groups presented higher corticosterone plasma levels (ng/ml) compared to C (2.0 ± 0.7) and EE (2.8 ± 0.9) respectively, and CMS+EE group showed lower plasma corticosterone in comparison with group CMS. In the FST, CMS (187 ± 14) presented higher immobility (s) compared with C (123 ± 9), EE (129 ± 14) and CMS + EE (138 ± 6) groups. In the Y‐maze test the difference scores of entries (%) were lower in the CMS (1 ± 3) and CMS + EE (16 ± 5) compared to C (15 ± 3) and EE (28 ± 5) respectively. EE and CMS + EE showed increased entries difference score compared with C and CMS respectively. EE reduced corticosterone release, canceled the depression‐like behavior and the impairment of memory induced by CMS showing its protective effect.
Mitogen‐inducible gene 6 potentiates glucolipotoxicity‐induced pancreatic beta cell death
Authors: Chen Yi‐Chun , Colvin E. Scott , Fueger Patrick T.
Published: June 2021

Destruction of the pancreatic insulin‐secreting beta cells, triggered by endoplasmic reticulum (ER) stress and glucolipotoxicity (GLT), is a common feature of type 2 diabetes. We previously identified that the endogenous epidermal growth factor inhibitor mitogen‐inducible gene 6 (Mig6) is induced by ER stress to promote beta cell apoptosis. In this study, we examined: 1) whether GLT also stimulates Mig6 expression, and 2) the cellular signaling cascades that interplay with Mig6 to facilitate GLT‐induced beta cell death. Using INS‐1‐derived 832/13 beta cells, we discovered that glucose, but not palmitate, stimulates Mig6 expression in a dose‐ and time‐dependent manner. Utilizing adenoviral‐mediated delivery of Mig6 to 832/13 cells, we found that Mig6 exacerbated GLT‐induced pro‐apoptotic JNK activation and caspase 3 activation. Furthermore, Mig6 might promote beta cell death by blocking cellular pro‐survival signaling pathways, as we have demonstrated that Mig6 overexpression inhibits the Akt and Erk signaling pathways in 832/13 cells. In conclusion, high glucose induces Mig6 expression in the pancreatic beta cells, which might facilitate GLT‐induced cell death. Supported by NIH DK078732(To PTF)
Influence of photoperiod in exercise performance and central nervous system activation
Authors: Machado Frederico Sander Mansur , Fóscolo Daniela , Niero Larissa , Coimbra Candido Celso
Published: June 2021

Aims

In the present study, we sought to investigate the relationship between photoperiodic daily fluctuations of internal body temperature (Tcore) with physical capacity (Workload) and neuronal activation induced by progressive treadmill exercise test.

Methods

Animals were subjected to protocols of progressive exercise test (n=11), resting on treadmill (n=12) or without prior manipulation (n=12) during dark and light phases. At the end of the last experimental session, the animals were perfused and the brains removed for immunohistchemical analysis of neuronal activation in the locus coeruleus (LC), paraventricular nucleus (PVN), supra‐optic nucleus (SO) and suprachiasmatic nucleus (SQN).

Results

Workload was 23% higher in the light phase when compared to dark phase exercise (p<0.001). PVN, SO and LC were strongly activated by exercise until fatigue independent of circadian phase (p<0.001). The SQN was not activated neither by exercise nor time of the day. A strong correlation between the activation of PVN, SO and LC was found only during the dark phase of the photoperiod.

Conclusion

Thereafter, the time of the day, possibly through its centrally mediated mechanisms favors physical performance during light phase independent of basal spontaneous locomotor activity levels. Besides, the SQN seemed to be resistant to exercise related disturbances.

Research sponsored by CAPES, CNPq and FAPEMIG.
Use of the Cam Kinase Kinase β Inhibitor is Contraindicated in Calcium Imaging Studies
Authors: Jurcsisn Jennifer , Pye Richard Laurence , Barr Barbara Lyn , Wyatt Christopher Neil
Published: June 2021

The energy‐sensing enzyme AMP‐activated protein kinase (AMPK) may play a critical role in oxygen‐sensing by the carotid bodies. To activate AMPK, it must be phosphorylated at Thr‐172 (alpha subunit) by upstream kinases. This study's objective was to determine the importance of CamKKβ in the hypoxic chemotransduction mechanism of oxygen‐sensing Type I cells within the mouse carotid body. Type I cells were loaded with Fura‐2 (5 μM). Intracellular Ca2+ was measured by exposing cells to 340nm and 380nm light and recording emission fluorescence at 510nm. Initial experiments were to see if Ca2+ increase during hypoxic exposure (10 Torr) could be altered by the CamKKβ inhibitor STO609 (100 μM). However, STO609 is green in solution and application caused large emission signal deviations. Faster changes in the 380nm signal caused a 340/380 ratio fall, suggesting intracellular Ca2+ decreased. Importantly, in the absence of cells, STO609 increased 510nm emission, indicating the inhibitor itself was fluorescing.

To avoid this artifact, cells were loaded with a ‘red emission’ Ca2+ indicator dye, x‐Rhod‐1 (5 μM), exposed to 580nm light, and emission was recorded at 602nm. Cells were excited with high K+ (50mM). During elevated Ca2+, STO609 (100 μM) was applied and an immediate fall in the emission signal was observed. This decrease in Ca2+ signal with STO609 was also seen in cells exposed to 0mM Ca2+ solutions containing ionomycin. Thus STO609 directly interacts with x‐Rhod‐1 and is therefore precluded in Ca2+ imaging studies.

Supported by NIH RO1‐HL091836
Ablation of sympathetic neurons projecting to mesenteric blood vessels attenuates nicotine induced rise in blood pressure (BP)
Authors: Hammond Bradley , Kreulen David
Published: June 2021

Celiac ganglionectomy attenuates the increase in BP seen in the DOCA‐salt model of hypertension; however, it is unknown which population of neurons in the celiac ganglion (CG) are responsible for this attenuation. Therefore, we ablated sympathetic post ganglionic neurons specifically innervating mesenteric blood vessels by applying a retrograde tracer, cholera toxin subunit b, conjugated to the neurotoxin saporin (CTB‐SAP) on two, adjacent branches of mesenteric arteries and veins. Control animals received PBS instead of a CTB‐SAP treatment. 1wk after CTB‐SAP application, rats were anesthetized, fitted with a femoral catheter, and the CG was dissected. Nicotine was then applied to the CG, which caused a significant increase in BP in control animals but not in CTB‐SAP treated animals. Immunohistochemistry of the CG revealed neuronal destruction in CTB‐SAP treated animals when compared to control animals. This study shows ablating CG neurons that innervate a small portion of the vasculature reduces the effect of CG stimulation on systemic BP, suggesting these neurons are likely to innervate a sufficient number of blood vessels to regulate systemic BP. Moreover, this study gives way for further investigations into the differing roles of separate neuronal sub populations in the CG. RC060365

Blood Pressure (mmHg)
Baseline
Nicotine (200 mM)

Control
74.6 ± 5.9
90.2 ± 6 *

CTB‐SAP
99.3 ± 19.8
94.3 ± 9.2 *

p ≤ 0.05 to baseline
AT1a Receptor Knockdown in the Median Preoptic Nucleus Attenuates the Sustained Component of Hypertension Resulting from Chronic Intermittent Hypoxia
Authors: Shell Brent , Little Joel , Cunningham Tom
Published: June 2021

Obstructive sleep apnea produces hypertension and increases sympathetic nerve activity during the hypoxic sleeping events as well as during the waking hours. To model the hypoxemia experienced during sleep apnea, rodents are exposed to chronic intermittent hypoxia (CIH). The median preoptic nucleus (MnPO) is a forebrain region that contributes to this sustained increase in mean arterial pressure (MAP) from CIH. This region integrates information from forebrain circumventricular organs and projects to the paraventricular nucleus to influence sympathetic nerve activity. Our lab has previously shown that in response to CIH there is increased ΔFosB expression in the MnPO and that after a 7 day hypoxia protocol MnPO AT1a receptor mRNA is increased 7 fold whereas AT1b shows no change. In this study, we tested the hypothesis that MnPO AT1a receptors contribute to CIH hypertension using adult male Sprague Dawley rats injected with a neuron specific adenoviral vector with either shRNA to knock down expression of AT1a receptors in the MnPO or a scrambled RNA sequence. All rats showed an increase in MAP during the hypoxic light period, but rats receiving the AT1a knock down did not exhibit the sustained increase in MAP during the normoxic dark phase (P<.05). This data indicates that the AT1a receptor in the MnPO is necessary for the sustained component of hypertension resulting from CIH. P01 HL‐88052
High‐Throughput Fluorescence Assay for Membrane‐Protein Interaction
Authors: Kim Hyunjin , Afsari Hamid Samareh , Sheng Ren , Cho Wonhwa
Published: June 2021

Membrane‐protein interactions play key roles in a wide variety of biological processes, including cell signaling. Although various methods have been employed to measure membrane binding of soluble proteins, a robust high‐throughput assay that is universally applicable to all proteins is lacking at present. Here we report a new fluorescence quenching assay utilizing enhanced green fluorescence protein (EGFP)‐fusion proteins and a lipid containing a dark quencher, N‐dimethylaminoazobenzenesulfonyl‐phosphatidylethanolamine (dabsyl‐PE). The EGFP fluorescence emission intensity showed a large decrease when EGFP‐fusion proteins bound the vesicles containing 5–10 mole% dabsyl‐PE. This simple assay, which can be performed using either a cuvette‐based spectrofluorometer or a fluorescence plate reader, allowed rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid‐binding domains, including two pleckstrin homology (PH) domains, an epsin N‐terminal homology (ENTH) domain, and a phox (PX) domain. The assay was also successfully applied to high‐throughput screening of small molecules that modulate membrane binding of proteins.
Interleukin‐1β upregulates components of the intra‐renal renin‐angiotensin system but does not induce salt‐sensitive hypertension.
Authors: Boesen Erika
Published: June 2021

Macrophage infiltration of the kidney is frequently observed in salt‐sensitive hypertension, but the role of these cells in blood pressure control is incompletely understood. We tested whether interleukin‐1β, a key pro‐inflammatory cytokine produced by macrophages, affects expression of components of the renin‐angiotensin system in the kidney, and whether chronic elevation of IL‐1β levels induces salt‐sensitive hypertension. Male C57BL/6 mice underwent 14‐day s.c. IL‐1β infusion (10ng/h), while maintained on either a normal (0.4% NaCl; n = 6) or high salt diet (4% NaCl; n = 6). Normal or high salt diet‐treated mice not undergoing IL‐1β infusion served as controls (n = 4 & 4). Quantitative real‐time PCR revealed that renal cortical expression of renin and angiotensinogen approximately doubled in both normal (2−ΔΔCT = 2.3 ± 0.4 and 1.9 ± 0.4) and high‐salt treated mice receiving IL‐1β infusion (2−ΔΔCT = 1.9 ± 0.5 and 1.9 ± 0.4) compared to non‐infused normal salt (2−ΔΔCT = 1.1 ± 0.2 and 1.1 ± 0.2) and high salt‐treated mice (2−ΔΔCT = 1.0 ± 0.1 and 1.1 ± 0.2; PInfusion < 0.05 for both genes). No significant difference in renal cortical angiotensin AT1a receptor expression was observed between groups. Systolic blood pressure (tail cuff plethysmography) at day 14 of IL‐1β infusion was not significantly different between normal (108 ± 5 mmHg) and high salt‐diet treated mice (105 ± 4 mmHg). Thus, despite increasing expression of key components of the renin‐angiotensin system, chronic elevation of IL‐1β does not induce salt‐sensitive hypertension.
Very low levels of H2S in the blood are needed to affect the medullary respiratory neurons and the arterial chemoreceptors in‐vivo
Authors: Klingerman Candice M. , Haouzi Philippe
Published: June 2021

Endogenous H2S has been proposed to be involved in the generation of breathing by the brainstem respiratory neurons (RN) and to transduce the effects of hypoxemia in the arterial chemoreceptors (CB). However, H2S at concentrations which should inhibit the cytochrome C oxidase (CCO) activity, i.e. above 30 μM, have been used in‐vitro to stimulate the CB (30–100 μM) or to affect the RN (200 μM). We determined the concentration of H2S dissolved in the arterial blood able to stimulate the CB and to stop breathing in 8 spontaneously‐breathing anesthetized rats. H2S partial pressure (PaH2S) and gaseous concentrations were determined in the arterial blood from alveolar PH2S during continuous venous infusion of NaHS. This approach overcomes most of the issues of other techniques that directly measure H2S dissolved in the blood. A stimulation of breathing occurred for a PaH2S of 0.007±0.003 mmHg, corresponding to a concentration of gaseous and total dissolved H2S of at most 0.63±0.31 μM and 1.89±0.93 μM. A rapid reduction in breathing occurred at a PaH2S of 0.042±0.020 mmHg, gaseous H2S of 3.68±1.72 μM, and total dissolved H2S of 11.04±5.16 μM. H2S affects the CB and RN in‐vivo via mechanisms independent of CCO. Whether these low levels of dissolved H2S are low enough to be compatible with the levels of H2S endogenously produced remains to be determined. Support provided by NIH grant 1R21NS080788–01.
Uncoupled H transport by the Na/K pumps expressed in Xenopus oocytes
Authors: Mitchell Travis J. , Olivera J. Fernando , Gatto Craig , Artigas Pablo
Published: June 2021

The Na/K pump (NKA) is a P‐type ATPase that maintains ion gradients across the plasma membrane of animal cells by exporting 3 Na in exchange for 2 K, per each ATP hydrolyzed. The molecular mechanism of ion selectivity at the binding sites is not fully understood. Two out of three ion binding sites bind both Na and K (shared sites) and another exclusively binds Na. Without external Na (Nao) or K (Ko) the pump transports H passively through a pathway and mechanism that is, as yet, uncharacterized (H‐leak). In order to better understand this atypical transport mode and the interaction of ions at the binding sites we investigated the characteristics of this H‐leak. We characterized external [H] dependence, inhibition by Nao and Ko and voltage dependence of NKA heterologously expressed in Xenopus oocytes via two electrode voltage clamp and giant patch clamp. Increasing [H]o increased inward current with a strongly V‐dependent K0.5 (0.4 μM at −180 mV and 1.3 μM at −80 mV). At pH ≤ 5.6, the rapid increase in current was followed by a slower current inhibition (80% inhibition at pH 5 in steady state). At pH 7.6 and 6, the leak was inhibited by Nao or Ko with a mild V‐dependence (K05 ≃ 10 mM and 0.1 mM, respectively at −180 mV). At pH 5, Na had a biphasic effect, increasing the current at < 5 mM and inhibiting at higher [Na], indicating relief of the H dependent inhibition. Supported by AHA BGIA2140172 to PA & NIH GM061583 to CG.
Discovering a sialic acid independent ligand for paired receptors Siglec‐5 and ‐14 (1003.5)
Authors: Fong Jerry , Deng Liwen , Varki Nissi , Nizet Victor , Varki Ajit
Published: June 2021

Siglec‐5 and Siglec‐14 are sialic acid (Sia)‐binding immunoreceptors found on myeloid lineage cells. Despite having identical ligand binding domains due to gene conversion, Siglec‐5 inhibits inflammatory responses after ligand engagement, but Siglec‐14 activates it instead. This unusual combination defines Siglec‐5 and ‐14 as paired receptors. Since sialoglycan ligands for Siglec‐5 are widely distributed on host cell surfaces, this is hypothesized to be a self‐recognition mechanism to prevent innate immune cells from attacking “self.” However, some bacterial pathogens take advantage of this self‐recognition system by engaging Siglec‐5, in order to suppress immune responses. Thus, it is hypothesized that Siglec‐14 emerged to counteract these “hijacking” pathogens. Although this model of Siglec‐14 evolution predicts uni‐directional gene conversion between Siglec‐5 and ‐14, the ligand binding domains of these receptors had undergone bi‐directional gene conversion instead. Also Siglec‐5 and ‐14 of some nonhuman primates have independently lost the ability to bind Sias due to a mutation in the Sia recognizing pocket. Thus, these receptors may have evolved to engage a Sia independent endogenous ligand. Preliminary evidence indicates that that such a ligand exists. Ongoing experiments are addressing whether this putative ligand‐receptor interaction modulates the pro‐inflammatory response.

Grant Funding Source: Supported by NHLBI P01 HL057345