bluuebunny/crossref_metadata_2025 huggingface dataset:
The small heart of the Ornate Tinamou is compatible with endothermy and flight but compromises aerobic metabolism and thermoregulation during recovery from exhaustive activity
Authors: Altimiras Jordi , Deck Lina Maria Giraldo , Garitano‐Zavala Álvaro
Published: June 2021

Tinamous are an ancient family of neotropical flying birds. Scant data from a few species show that they have a small heart so our aim was to characterize relative heart size of two species of the genus Nothoprocta and assess the physiological limitations associated with a small heart size. Relative heart size (0.24% for the Ornate Tinamou OT, a highland species and 0.28% for the Chilean Tinamou CT, a lowland species) was significantly smaller than high and lowland chickens (0.54% and 0.42% respectively), without evidence of right ventricular hypertrophy. Resting aerobic metabolism was 31% lower in OT than in highland chickens. When subjected to exhaustive activity, OT had elevated glucose and lactate levels suggesting a severe oxygen debt when exhausted. This was further shown as a significant drop in body temperature after an exhaustive bout. Finally heart rate while running on a treadmill at 3 km h−1 was 5% lower in OT, indicating that tinamous cannot compensate for the reduction in heart size with a faster heart rate. Altogether, we provide evidence that heart size is a phylogenetically conserved trait among tinamous and that the Ornate Tinamou cannot compensate aerobically for its small heart. Instead, it relies on anaerobic metabolism incurring in a large oxygen debt while exhausted. Supported by FORMAS Centre of Excellence in Animal Welfare Science and career grant from Linköpings universitet to JA.
Using corrosion casts to understand oxygen delivery in the grasshopper leg
Authors: Ferrara Nicholas M. , Kirkton Scott D.
Published: June 2021

The light‐weight, air‐filled tracheal system has allowed insects to be the most evolutionary successful animals on the planet. Oxygen enters through spiracular openings before passing down tracheal tubes that branch into successively smaller tubes that eventually become tracheoles, which serve as the site of gas exchange. Depending on the species and life history stage, oxygen delivery down the tracheae may occur via diffusion, convection, or both. While adult Schistocerca americana grasshoppers use abdominal pumping and convection to drive oxygen through their head, thorax, and abdomen, it is less clear how oxygen delivery occurs down the metathoracic leg. We cannulated the tracheal system of an adult grasshopper and injected a low viscosity resin under vacuum, to create a corrosion cast of the major tracheal branches of the metathoracic leg. After the low viscosity resin had solidified, grasshopper tissue was digested leaving only the tracheal cast. Using light microscopy, we measured the radii of the main tracheae and their subsequent branches. These tracheal dimensions followed Murray's Law, suggesting that the network provided convective oxygen delivery. We will examine the casts using scanning electron microscopy to visualize tracheal branching patterns and estimate oxygen flow patterns throughout the metathoracic leg. This research has been supported by funding from Union College.
Specific and Progressive Diaphragmatic Myopathy During the Development of Heart Failure
Authors: Foster Andrew James , Arkell Alicia , Platt Mathew , Simpson Jeremy A. , Murrant Coral L.
Published: June 2021

Respiratory dysfunction is a prominent feature of heart failure (HF) but remains to be fully understood. Our objective was to characterize diaphragmatic myopathy that manifests during the development of HF.

Methods

HF was induced in mice by transaortic constriction (TAC). Cardiorespiratory function was measured in vivo and diaphragm strips were used for histology and in vitro contractile function in mice at 2, 4 or 18 weeks TAC or in sham mice.

Results

In vivo respiratory frequency and pressure did not differ at any time point in anaesthetized animals suggesting absence of respiratory workload. In vitro force production was significantly elevated at 2 weeks (p<0.05) with a similar trend at 4 weeks; by 18 weeks, force production returned to sham levels. Indices of contractile performance (peak tension/time & dF/dt) at 2 and 4 weeks were significantly increased (p<0.05) and returned to sham levels by 18 weeks. Fatiguing stimulations revealed impaired relaxation at both 2 and 4 weeks TAC (p<0.05). Diaphragmatic atrophy (reduced cross sectional area) and fibrosis were evident at 4 weeks (p<0.05) and continued to develop by 18 weeks.

Conclusions

Diaphragmatic myopathy develops in the absence of increased workload and is demonstrated by contractile alterations, atrophy and fibrosis.
PKCtheta Contributes to Myotube Formation by Regulating Protein Synthesis
Authors: Marino Joseph S , Hinds Terry , Hoover Rachael A , Ondrus Eric , Onion Jeremy L , Dowling Abigail , McLoughlin Thomas , Sanchez Edwin R , Hill Jennifer W
Published: June 2021

The insulin signaling cascade regulates skeletal muscle protein synthesis. Protein kinase C theta (PKCθ) negatively regulates the insulin signaling cascade. Here we determine the role of PKCθ in myotube formation, a process critical to muscle development, regeneration, and hypertrophy. Using sh‐RNA, we established C2C12 cells with 75% reduction in PKCθ (PKCθKD). PKCθKD myoblasts had enhanced fusion and myotube density, and increased protein synthesis following 4 days of differentiation. Phosphorylation of IRS1ser1101, a PKCθ inhibitory target, was reduced in PKCθKD 4 day old myotubes. Phosphorylation of IRS1ser636/639, substrates of ERK which promote IRS1 degradation, was increased in PKCθKD myotubes and supported by increased ERK activation. Surprisingly, insulin receptor (IR) tyrosine phosphorylation was reduced in PKCθKD myotubes, suggesting insulin resistance. However, PKCθKD and control myotubes both responded to insulin by increasing AKTser473 phosphorylation indicating insulin sensitivity. These findings suggest that reduced IR tyrosine and increased IRS1 serine phosphorylation in PKCθKD myotubes may be the result of protein synthesis negative feedback. Taken together, PKCθ contributes to the events of myotube formation by regulating protein synthesis. Future work is warranted to pursue PKCθ as a viable target to enhance skeletal muscle regeneration and hypertrophy in vivo.
Satellite cells regulate the skeletal muscle environment by inhibiting fibroblast function
Authors: Fry Christopher S , Lee Jonah D , Jackson Janna R , Kirby Tyler J , Dupont‐Versteegden Esther E , McCarthy John J , Peterson Charlotte A
Published: June 2021

Interaction between satellite cells and the skeletal muscle extracellular matrix (ECM) has been demonstrated, but a role for satellite cells in regulating the ECM during hypertrophy has not been defined. We utilized mouse primary myoblasts and fibroblasts in co‐culture in addition to a genetically modified mouse model (Pax7‐DTA) to investigate the role of satellite cells in the regulation of muscle ECM. The Pax7‐DTA mouse allows for the conditional depletion of satellite cells in adult muscle following tamoxifen administration. Vehicle/tamoxifen treated animals were randomized to sham or synergist ablation surgery, leading to overload of the plantaris muscle for 1 and 8 weeks. Microarray analysis after 1 week of surgery showed the loss of satellite cells was associated with increased expression of numerous collagens and ECM structural genes. Depletion of satellite cells also led to attenuated hypertrophy and fibrosis of the plantaris with increases in both ECM deposition and fibroblast number following 8 weeks of overload. Co‐culturing mouse myoblasts with fibroblasts led to decreased mRNA expression of Col Ia2, IIIa1, VIa2 and Fibronectin compared to fibroblasts cocultured with fibroblasts, suggesting a paracrine role of myoblasts that inhibits fibroblast ECM production. We conclude that a novel function of satellite cells both at rest and during hypertrophy is to regulate muscle fibroblasts and the ECM.

Funding: NIAMS R01AR060701, R21AG34453 and the Jeane B. Kempner Postdoctoral Award.
Cardiovascular and metabolic regulation in mice with neuron specific deletion of the leptin receptor.
Authors: do Carmo Jussara Marcia , da Silva Alexandre A. , Pace Benjamin R. , Davis Mark T. , Hall John E.
Published: June 2021

We showed that male leptin‐deficient mice do not develop hypertension despite severe metabolic abnormalities. We examined the impact of CNS leptin receptor (LR) deletion on energy balance and obesity‐induced elevations in blood pressure (BP) and heart rate (HR). BP and HR measured by telemetry, and motor activity (MA) and oxygen consumption (VO2) were monitored for 5 days in 24‐week‐old female and male LR/Nestin‐Cre (n=6/gender) and control mice (WT, n=5/gender). Female and male LR/Nestin‐Cre mice were heavier (60„b4 and 62±5 vs 24±1 g and 31±1 g) due to hyperphagia (5.2±0.5 and 6.2±0.5 vs 3.8±0.5 and 3.5±1.0 g/day) and reduced VO2 (30±2 and 27±1 vs 50±8 and 49±3 ml/kg/min) and MA (16±3 and 3±1 vs 507±80 and 676±105 cm/hr) compared to WT mice. LR/Nestin‐Cre mice were hyperleptinemic (109±2 vs 7±1 ng/ml) and hyperinsulinemic (119±23 vs 10±3 μU/ml), but had normal glucose levels (174±10 vs 180±11 mg/dl) compared to WT mice. Female LR/Nestin‐Cre mice exhibited higher BP than WT mice (117±2 vs 110±2 mmHg); BP in male LR/Nestin‐Cre was not different than in WT mice (110±1 vs 111±3 mmHg). HR was slightly lower in female and male LR/Nestin‐Cre mice (617±14 and 580±10) vs WT mice male and female mice (648±12 and 650¡Ó35 bpm). These results highlight the importance of CNS LRs in regulating appetite and energy expenditure and show potential gender differences in the impact of obesity on control of BP. (NHLBI‐PO1HL51971/AHA SDG5680016)
Brain‐derived neurotrophic factor augments the contraction of intestinal longitudinal muscle strips induced by Carbachol
Authors: Alqudah Mohammad , Mahavadi Sunila , Srinivasa Karnam M. , Grider Jack
Published: June 2021

Background

We have demonstrated that intestinal longitudinal smooth muscle cells synthesize and secrete brain derived neurotrophic factor (BDNF) in response to neuropeptide transmitters. But the function of this BDNF in the gastrointestinal tract is undetermined.

Aim

to test the effect of BDNF on agonist‐induced contraction in intestinal longitudinal smooth muscle.

Material and method

Rabbit longitudinal muscle strips were prepared from the intestine. The contraction of the muscle strips was recorded in response to substance P 10–6M (SP 10–6) and Carbachol 10–5M (CCh 10–5). Muscle strips were preincubated with 10 nM exogenous BDNF and the changes in SP/CCh‐induced contraction were examined. A Trk antagonist and two Trk B specific agonists were used to investigate the role of the Trk B receptor in the effect of BDNF on contraction.

Results

BDNF significantly enhanced the contraction of longitudinal smooth muscle induced by CCh 10–5. However, contraction induced by SP 10–6 was not affected by pretreatment with BDNF. The pan Trk receptor inhibitor (K252a) inhibited the effect of BDNF on CCh‐induced contraction. Moreover, two Trk B agonists (LM 22A4 & 7, 8‐dihydroxyflavone) augmented the CCh‐induced contraction of the longitudinal muscle strips.

Conclusion

BDNF enhances the cholinergic‐induced longitudinal muscle contraction through the activation of its cognate Trk B receptor.
Dopamine enhances gastric motility through D1 dopaminergic receptors on cholinergic neurons in the myenteric plexus of rats
Authors: Song Jin , Zheng Lifei , Xu Jingdong , Zhu Jinxia
Published: June 2021

Objectives

Investigate the effect of dopamine on the gastric motility and the underlying mechanism.

Methods

Immunofluorescence and gastric motility recording by organ bath study.

Result

The immunoreactivities of D1 and D2 were distributed in the choline acetyltransferase (ChAT)‐immunoreactive neurons in the myenteric plexus of rats. The gastric motility in vitro was enhanced from 8.2 to 10.8£g¡Ámin£©by DA in the concentrations of 1¡Á10–8mol/L (n=10, p<0.05). However, DA manifested inhibitory role at the higher concentrations of 3¡Á10–6mol/L. The excitatory effect of DA was mimicked by SKF‐38393, a selective agonist of D1‐like receptors in dose‐dependent manner, and blocked by neuronal sodium channel blocker, tetrodotoxin (TTX) in 1¡Á10–5mol/L and atropine (competitive nonselective antagonist of muscarinic acetylcholine receptors) in 1¡Á10–5mol/L (n=8, p<0.05). The selective D2‐like receptor antagonist sulpride and selective D2‐like agonist Quipirole did not affect both the excitatory and inhibitory effect of DA.

Conclusions

The present study demonstrates that dopamine is able to enhance gastric motility in lower concentration through D1 dopaminergic receptors activating acetylcholine release from myenteric nerve plexus. The higher dose of DA can inhibit gastric motility, but the mechanism needs to be further investigated.
Presence of luminal acetate sensing and absorption mechanisms in rat duodenum
Authors: Kaji Izumi , Akiba Yasutada , Kaunitz Jonathan D
Published: June 2021

Duodenal lumen short‐chain fatty acids (SCFA) are derived from ingested food or bacteria. Since SCFA receptors GPR41 and 43 are expressed on duodenal endocrine cells, and since a selective GPR43 agonist increases duodenal HCO3− secretion (DBS) via muscarinic/5‐HT4 receptor activation, we further examined the effect of luminal acetate on DBS. We measured DBS with pH and CO2 electrodes in a perfused duodenal loop in anesthetized rats. GPR41 colocalized with glucagon‐like peptide (GLP)‐1, whereas GPR43 colocalized with 5‐HT. Luminal perfusion of acetate dose‐dependently increased DBS with increasing GLP‐2 release. Acetate‐induced DBS was reduced by the 5‐HT4 receptor antagonist GR113808, pretreatment with indomethacin or high‐dose capsaicin, or co‐perfusion of the monocarboxylate transporter (MCT) inhibitor 4‐CHCA. Na+‐coupled MCT‐1 (SMCT1) was localized on the brush border and MCT4 on the basolateral membrane of upper villous epithelial cells, whereas MCT1 was expressed on the basolateral membrane of lower villous and crypt cells. Feeding increased the membrane localization of MCT1 and 4 with increased mRNA expression, compared to overnight fasting. Luminal acetate‐induced DBS via GLP‐2, 5‐HT, prostaglandin and capsaicin pathways, and acetate absorption via SMCT1 and MCT, may be implicated in the pathogenesis of functional dyspepsia and bacterial overgrowth. VA Merit Review, NIH‐NIDDK R01 DK54221
Increased corticosterone levels contribute to glucose intolerance induced by the absence of melatonin
Authors: Rodrigues Sandra Campos , Pantaleão Lucas , Lellis‐Santos Camilo , Veras Katherine , Amaral Fernanda , Anhê Gabriel , Bordin Silvana
Published: June 2021

Pinealectomized rats (PINX) become glucose intolerant and exhibit increased gluconeogenesis. Melatonin is known to reduce glucocorticoid (GC) secretion, but the role of GC on the glucose intolerance of PINX is not described. Here we evaluated the participation of GC in the glucose intolerance of PINX rats. Rats were subjected to pinealectomy or fictitious surgery (sham) and treated with RU486 [0,5mg/100g/5days] or vehicle 25 days after surgeries. After that, rats were subjected to glucose and pyruvate tolerance tests. Livers were used for assessment of insulin‐induced maximal AKT activation; plasma was collected for insulin and corticosterone. The experiments were conducted at the end of the light phase of the light/dark cycle. Corticosterone levels were 30% higher in PINX (p>;0.05), compared to sham. Glucose intolerance and upregulated gluconeogenesis found in PINX were reduced by 50% after treatment with RU486 (p>;0.05). Reduced insulin levels found in PINX rats were recovered by RU486. Concordantly, insulin‐induced AKT phosphorylation in PINX (that was decreased when compared to sham; p>;0.05) was also upregulated after treatment with RU486 by 40%. We concluded that glucose intolerance of PINX rats is characterized by hepatic insulin resistance and reduced insulin levels. These metabolic adaptations may be a result of increased corticosterone levels.

Financial Support: FAPESP (2011/12329–1)
Luminal acid‐associated post‐prandial epithelial hypoxia maintains the expression of iron absorptive DMT1 and Dcytb in rat duodenum
Authors: Akiba Yasutada , Kaji Izumi , Kaunitz Jonathan D
Published: June 2021

The duodenal mucosa, cyclically exposed to gastric acid, absorbs dietary iron via duodenal cytochrome b reductase (Dcytb) and the divalent metal ion transporter‐1 (DMT1), upregulated by hypoxia inducible factor‐2α. Since acid increases luminal H2O2 production via the ATP‐P2Y‐Duox2 pathway, we hypothesized that luminal acid induces epithelial hypoxia, affecting the expression of Dcytb and DMT1. We measured epithelial hypoxia by Hypoxyprobe staining on cryostat sections. Pimonidazole was given ip to overnight fasted, freely fed, or omeprazole (OPZ)‐ or catalase (Cat)‐treated rats. The duodenal loop of anesthetized rats was perfused with pH 7 or pH 2.2 saline for 10 min, following pimonidazole iv. Hypoxyprobe staining was heterogeneous in duodenal villous cells: fluorescence intensity was higher in the duodenal villi of fed rats compared to fasting, luminal acid exposure increased Hypoxyprobe staining, compared to pH 7, and Hypoxyprobe staining was decreased in the duodenal villi of OPZ‐and Cat‐treated fed rats. Expression of DMT1 and Dcytb was doubled in the fed group, compared to the fasted group, and inhibited by OPZ treatment. Acid exposure with H2O2 production may induce post‐prandial pO2 fluctuation and maintain the expression level of DMT1 and Dcytb, implicated in OPZ‐induced iron deficiency. VA Merit Review, NIH R01 DK54221
Changes in angiotensin II‐induced vasomotor function from newborn to senescence: correlation with expression of AT1 and AT2 receptors
Authors: Vamos Zoltan , Cseplo P , Batai Z , Torok O , Ivic I , Matics R , Hamar J , Koller A
Published: June 2021

Angiotensin II (Ang II) via activating angiotensin type 1 receptors (AT1R) is a potent vasoconstrictor, whereas recent studies suggest a role for AT2R in the modulation of vasomotor tone (VT). There are few data, however regarding aging‐induced changes in Ang II‐induced VT. We hypothesized that from newborn to senescence age there are substantial changes in the Ang II‐induced VT, mediated by AT1R and/or AT2R.

Changes in isometric force to Ang II of carotid arteries (CA) of newborn (8 days: 8d), young (2–9 months[m]: 2–9m), adult (12–20m) and senescence (20–30m) rats were measured with a wire myograph (DMT 610). To assess the role of AT2R, an AT1R blocker (losartan) was used. Expressions (exp.) of AT1R and AT2R of CA were determined by rt‐PCR.

Ang II‐induced contractions increased from 8d to 6m (8d: 1.4±0.4, 1m: 2.3±0.2, 2m: 5.3±0.5, 6m: 5.9±0.4 mN), then decreased to the age of 30m (9m: 5.5±0.2, 12m: 5.2±0.5, 24 m: 4.8±0.2, 30m: 3.7±0.6mN,). In the presence of losartan, Ang II did not elicited significant changes VT in any age group. Exp. of AT1R correlated with functional data: increased from 8d to 12m (8d: 1±0, 6m: 12±3.5, 12m: 15±2.7 cpm), then decreased to the age of 30m (18m: 7.3±0.5, 30m: 2.4±0,8cpm). There were no differences in the exp. of AT2R form 8d to 30m.

In conclusion, Ang II‐induced vasoconstriction via AT1R showed an inverse U‐shape as a function of age. The similar pattern in vascular AT1R exp. suggests genetic determination.

Supported by HNSRF ‐ OTKA K71591 and T67984, AHA F. Aff., 0555897T, HSH 2010/2011 and SROP‐4.2.1/b‐10/2/KONV‐2010–0012, SROP‐4.2.2.A‐11/1/KONV‐2012–0024.
Administration of PNS on collagen level in tissue of lung fibrosis in mice
Authors: Xiaofang SUN , NIU Jianzaho , DUAN Fei , HUANG Chunfang , WANG Jifeng
Published: June 2021

To study the administration PNS(Panax notoginsenosides) on collagen level in lung tissue for mice. One hundred and eighty C57BL/6 male mice were divided into six groups: sham group, model group, PNS high, medium and low dose group, prednisone group (n = 30). In addition to the sham group, the remaining mice received a single intratracheal instillation of bleomycin, sham group received a single intratracheal instillation with saline. Two days after modeling, starting administration of PNS, until the day before the animals were killed. Administration of PNS high, medium and low dose group were 120, 60, 30mg/kg/d respectively, administration of prednisone group was 0.56mg/kg, sham group, model group were given equal volume of saline. All the animals at 7, 14, 28 d were killed, lungs were taken for HE, Mallory staining, and determination of lung hydroxyproline content.

RESULTS

Administration of PNS could reduce pulmonary fibrosis in mice as indicated by lower collagen content of lung tissue, reduce the proliferation of fibrous lung tissue.

Conclution

Administration of PNS can reduce pulmonary fibrosis in mice with less collagen content of lung tissue in mice. Supported by 111 project B07007
Pro‐inflammatory Role of Beta2‐Adrenoceptor Signaling in Human Airway Epithelial Cells in Response to IL‐13
Authors: Al‐Sawalha Nour Ahmad , Pokkunuri Indira , Bond Richard A. , Knoll Brian J.
Published: June 2021

We have shown the role of β2‐adrenoceptor (β2AR) signaling in inducing mucin content and inflammation in murine model of asthma. Our objective was to study the role of β2AR signaling in chemokine production and expression of a major airway mucin coding gene, MUC5AC, in normal human bronchial epithelial cells (NHBECs) in response to IL‐13. NHBECs were grown at air‐liquid interface (ALI) on transwell filters in the presence of 3μM epinephrine. Chemokine levels in subnatants were measured by multiplex ELISA on day 5 ALI. MUC5AC expression and phospho‐ERK1/2 were assessed by qRT‐PCR and immunoblotting on day 14 ALI. IL‐13 induced the release of eotaxin‐2, CXCL‐1 and MCP‐1 (eosinophil, monocyte, dendritic and memory T‐cell chemoattractants respectively) by 6.8, 3.8 and 6.9 fold respectively. Treatment with βAR antagonist nadolol reduced the levels of these chemokines to baseline. Nadolol and ICI‐118,551 (a β2AR selective antagonist) blocked IL‐13 induced MUC5AC expression, while CGP‐20712A (a β1AR selective antagonist) did not. U0126 (a MEK‐1/2 inhibitor) and H89 (a PKA inhibitor) reduced IL‐13 induced MUC5AC expression, but only U0126 reduced phospho‐ERK1/2 levels. Our data provides a strong evidence of the pro‐inflammatory role of β2AR signaling in mediating IL‐13 effect on human airway epithelium, and that ERK‐1/2 and PKA are components of the required β2AR signaling pathways.

Supported by NIH 1R01A179236 to RAB
Imidacloprid induces insulin resistance by protein kinase B (PKB) mediated mechanism
Authors: Kim Jonggun , Park Yooheon , Yoon Kyung Sup , Clark John M , Park Yeonhwa
Published: June 2021

Imidacloprid is one of the newest generations of insecticides, neonicotinoids, and is commonly used in crop protection and animal health. Recently, the correlation between imidacloprid and increased blood glucose levels in rats was reported. Thus, the purpose of the study was to determine effects of imidacloprid on glucose metabolism. Three difference cell models were used, adipocytes (3T3L1), hepatocytes (HepG2), and myotubes (C2C12). These cells were treated with imidacloprid (0, 10, and 20 μM) for 6–8 days followed by treatment with 10 nM insulin for 10 min to determine responses. Insulin stimulated glucose uptake was reduced by imidacloprid in all three cell culture models. Phosphorylation of Protein Kinase B (PKB), one of the major regulators of insulin signaling, was reduced without changing overall PKB expression by imidacloprid treatment. Subsequently, imidaclprid reduced phosphorylation of ribosomal S6 kinase (S6K), which is one downstream target of PKB and also feed‐back inhibitor of insulin signaling. These results suggest that imidacloprid could induce insulin resistance by affecting insulin signaling cascade, particularly up‐stream of PKB, in the adipocyte, liver, and muscle.
Pregnane X Receptor Mediates Dyslipidemia Induced by the HIV Protease Inhibitor Amprenavir in Mice
Authors: Helsley Robert Nathaniel , Sui Yipeng , Ai Ni , Park Se‐Hyung , Welsh William J. , Zhou Changcheng
Published: June 2021

HIV protease inhibitors have been used successfully in extending the lifespan of people infected with HIV. However, the mechanisms responsible for the increased risk of dyslipidemia and cardiovascular diseases associated with the use of protease inhibitors remain elusive. Several protease inhibitors have been implicated to activate the nuclear receptor pregnane X receptor (PXR), which acts as a xenobiotic sensor to regulate xenobiotic metabolism. We recently revealed PXR's pro‐atherogenic effects in animal models and found that chronic activation of PXR induces hyperlipidemia in wild‐type mice. In the present study, we found that a widely used HIV protease inhibitor, amprenavir, is a potent agonist of PXR. Amprenavir can induce expression of PXR target genes in human hepatoma HepaRG cells and intestinal LS180 cells. In silico ligand‐PXR docking studies combined with site‐direct mutagenesis identified several key residues within PXR's ligand binding pocket that constitute points of interaction with amprenavir. Lastly, amprenvir‐induced PXR activation significantly increased atherogenic lipoprotein LDL cholesterol levels in wild‐type mice, but had no effect in PXR knockout mice. These findings provide critical mechanistic insight for understanding the impact of protease inhibitors on cardiovascular disease and demonstrate a potential role of PXR in mediating adverse effects of HIV protease inhibitors in humans.

This work is supported by NIH grants T32HL072743, P30HL101300, P20GM103527 and AHA grant 09SDG2150176.
Non‐Canonical Signaling by GPCR‐Arrestin‐Gβγ Ternary Complexes
Authors: Wehbi Vanessa L , Feinstein Timothy Nathan , Vilardaga Jean‐Pierre
Published: June 2021

The classical model of arrestin‐mediated desensitization of GPCR is thought to be universal. In the current canonical model of GPCR signaling, arrestins terminate receptor signaling by impairing receptor–G protein coupling and promoting receptor internalization. However, this paradigm is incompatible with recent reports that the parathyroid hormone receptor (PTHR), a crucial GPCR for bone biology, sustains cAMP response for prolonged periods after ligand washout. Arrestins prolong GS‐mediated cAMP generation triggered by PTH, a process that correlates with the persistence of arrestin–PTHR complexes on endosomes, and which is thought to be associated with prolonged physiological calcemic and phosphate responses. This presents an inescapable paradox for the canonical model of arrestin‐mediated receptor–G protein decoupling. Here we show that PTHR forms a ternary complex that includes arrestin and Gβγ in response to PTH stimulation, which in turn increases the steady‐state levels of activated GS, leading to prolonged generation of cAMP. The non‐canonical signaling is not unique to the PTHR since we found that arrestin binding to the vasopressin V2 receptor prolonged cAMP production by vasopressin, which ultimately sustained water transport in kidney cells. This work provides a new model of GPCR signaling in which arrestins contribute to sustain agonist actions.

Supported by the NIH award R01DK087688.
Rab26 mediates the Golgi‐to‐cell surface transport of α2‐adrenergic receptors
Authors: Li Chunman , Fan Yi , Lan Tien‐Hung , Lambert Nevin , Wu Guangyu
Published: June 2021

The molecular mechanisms underlying the transport from the Golgi to the cell surface of G protein‐coupled receptors (GPCRs) remain poorly elucidated. Here we determined the role of Rab26, a Ras‐like small GTPase involved in vesicle‐mediated secretion, in the cell‐surface export of α2‐adrenergic receptors (α2‐ARs). We found that transient expression of Rab26 mutants and siRNA‐mediated depletion of Rab26 significantly attenuated the cell‐surface numbers of α2A‐AR and α2B‐AR as well as ERK1/2 activation by α2B‐AR. Furthermore, the receptors were extensively arrested in the Golgi by Rab26 mutants and siRNA. Moreover, Rab26 directly and activation‐dependently interacted with α2BAR, specifically the third intracellular loop. These data demonstrate that the small GTPase Rab26 regulates the Golgi‐to‐cell surface traffic of α2‐ARs, likely through a physical interaction. These data also provide the first evidence implicating an important function of Rab26 in coordinating plasma membrane protein transport (R01GM076167, R01GM096762 and R01GM078319).
Phosphorylation and internalization of short splicing variant of the omega 3 fatty acid sensor, GPR120
Authors: Sanchez‐Reyes Omar Benoni , Castillo‐Badillo Jean A. , Romero‐Avila M. Teresa , Takei Yoshinori , Hirasawa Akira , Tsujimoto Gozoh , Villalobos‐Molina Rafael , García‐Sáinz J. Adolfo
Published: June 2021

GPR120 is now recognized as an important therapeutic target for diabetes, due to of its anti‐inflammatory and insulin‐sensitizing effects after activation with Ω3 fatty acids. Phosphorylation of both receptor splicing variants dependent of Ω3 fatty acids, DHA and α‐LA, has been previously reported, nevertheless protein kinases involved in this regulation, as well as the specific sites of phosphorylation are not known. Using HEK 293 Flp‐In T‐REx cells stably expressing GPR120S‐Venus fusion protein we found phorbol ester PMA dependent phosphorylation of the receptor peaking after 30 min of stimulus in contrast of DHA and α‐LA induced phosphorylation peaking after 5 min of stimulus. A GPR120S‐Venus cell line also expressing α‐1B‐DsRed receptor construct was generated in order to depict receptor internalization as evidence of receptor crosstalk. Preliminary results show that after NA stimulus both receptors internalize, nonetheless after DHA stimulus only GPR120 internalize. PMA also induced both receptors to internalize. These results hint PKC as a kinase participating in heterologous regulation of GPR120S. Research sponsored by CONACyT (177556) and DGAPA (200812–3) grants.
A role for Endothelin Converting Enzyme‐1 activity in the regulation of neuromedin U receptor re‐sensitization and ERK signaling
Authors: Alhosaini Khaled , Challiss R.A.J , Willars G.B
Published: June 2021

The neuropeptide, neuromedin U (NmU) signals via two GPCRs, NMU1 and NMU2. NmU binding is pseudo‐irreversible leading to ligand/receptor internalization. Many GPCRs internalize after phosphorylation and arrestin binding, which desensitize G‐protein signaling but may promote G‐protein‐independent signaling (e.g. by scaffolding ERK pathway components). For some peptide ligands, endosomal proteolysis by endothelin converting enzyme‐1 (ECE‐1) enhances receptor recycling, perhaps by limiting ligand‐receptor interaction. This may influence receptor re‐sensitization and G protein‐independent events. Here we examined NmU/NMU2 internalization and roles of ECE‐1 in re‐sensitization of NMU2 and the duration of ERK activity. Data show internalization of NmU/NMU2 and desensitization of NMU2 Ca2+ responses by NmU‐25 that took 6h to re‐sensitize. Using pharmacological tools we show that dynamin‐dependent internalization and endosomal acidification but not protein synthesis promote re‐sensitization of NMU2 Ca2+ signaling. Furthermore, ECE‐1 activity promotes re‐sensitization and limits the duration of ERK signaling, consistent with NmU proteolysis enhancing NMU2 recycling but limiting G‐protein‐independent events.
Effects of Parabolic Flight on Serotonin‐related Gene Expression in the Mouse Brain
Authors: Yoshioka Mitsuhiro , Yamaguchi Taku , Ohta Hisashi , Ochiai Toshimasa
Published: June 2021

Exposure to uncontrollable stress triggers a wide range of adaptive changes in the CNS including the elevation of serotonin metabolism and an increased susceptibility to affective disorders. The purpose of the present experiment is to elucidate whether gene expression levels, especially serotonin‐related genes, are altered in the mouse brain exposed to gravity‐changing stress using a competitive reverse transcription‐polymerase chain reaction. Mice were exposed to gravity‐changing stress during 8 times repeated by parabolic flight. Serotonin and tryptophan transporter, and tryptophan hydroxylase‐2 mRNA levels in the midbrain 6 h after the flight were significantly increased compared with pre‐parabolic flight control. In contrast, those of 5‐HT1A receptor, GAD65/67, and tyrosine hydroxylase were not altered by the flight. These results suggest that the serotonergic system, particularly presynaptically synthetic pathway, might be activated in the CNS by gravity‐changing stress.
AAV2/1‐mediated gene delivery of CD200 into the hippocampus enhances neurogenesis and amyloid clearance in the APP mouse
Authors: Varnum Megan M , Kiyota Tomomi , Ingraham Kaitlin , Ikezu Seiko , Ikezu Tsuneya
Published: June 2021

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive decline of cognitive function and memory formation. Up‐regulation of inflammatory cytokines, microglial activation, and toxic Aβ in the AD brain negatively affect the ability of neural stem cells to mature and survive. CD200 is an anti‐inflammatory glycoprotein expressed in many cell types, including neurons, and its receptor is expressed in myeloid cells, like microglia. It has been shown in AD patients and mouse models that an age‐related increase in microglial activation is accompanied by an age‐related or Aβ‐induced decrease in CD200. We hypothesize that chronic neuroinflammation in AD contributes to suppression of neurogenesis in the hippocampus, and that this suppression can be restored by gene delivery of CD200. In this study the AD transgenic (Tg) mouse model expressing an APP mutant was intrahippocampally injected with adeno‐associated virus expressing CD200 at the pre‐symptomatic disease stage. Following treatment with CD200, mice were assessed for Aβ load, and neurogenesis was measured. CD200 expression enhanced proliferation and differentiation of neural stem cells in the dentate gyrus and reduced Aβ load. These data indicate that CD200 may be able to delay the onset of AD‐like pathology and decrease in neurogenesis, thus giving it potential as a therapeutic against neurodegeneration. NIH 5T32GM008541 to MMV.
Identification of brain‐targeted bioactive dietary quercetin‐3‐O‐glucuronide as a novel intervention for Alzheimer's disease
Authors: Pasinetti Giulio Maria
Published: June 2021

Previous studies indicate polyphenol intake from moderate consumption of red wines may lower relative risk of developing Alzheimer's disease (AD). We assessed accumulations of polyphenols in rat brain following oral dosage with Cabernet Sauvignon and tested brain‐targeted polyphenols for potential beneficial AD disease‐modifying activities. We demonstrated that the brain‐targeted polyphenol metabolite quercetin‐3‐O‐glucuronide significantly reduced generation of β‐amyloid (Aβ) peptides compared to control by primary neuron cultures from the Tg2576 AD mouse model. Using photo‐induced cross‐lining of unmodified proteins (PICUP) technique in vitro, we found quercetin‐3‐O‐glucuronide capable of interfering with the initial protein‐protein interaction of Aβ1–40 and Aβ1–42, necessary for the formation of neurotoxic oligomeric Aβ species. Quercetin‐3‐O‐glucuronide treatment significantly improved AD‐type deficits in hippocampal formation, basal synaptic transmission, and long‐term potentiation. Brain‐targeted quercetin‐3‐O‐glucuronide may modulate multiple independent AD disease modifying mechanisms and thus may contribute to benefits of dietary supplementation with red wines as an effective intervention for AD.
Cleavage of ROCK II: As a Common Phenomenon in Cell Death
Authors: Ozdemir Aysun , Ibisoglu Burcin , Simay Yaprak Dilber , Yaren Biljana Mitrovic , Ark Mustafa
Published: June 2021

We have previously showed that caspase‐2 cleaved Rho kinase II (Rock II), one of the Rho kinase isoforms and produced a novel ~140 kDa fragment of the enzyme in HUVEC during ouabain‐induced apoptosis. In the current work we evaluated whether this cleavage is a common process in cell death. To test this hypothesis two different types of cancer cell lines HeLa and HepG2 were exposed to two different cardiac glycosides (1μM, 18h); ouabain, digoxin and a standard chemotherapeutic; paclitaxel (1 μM, 18 h) to induce cell death. After the induction, the effect of Rock inhibitor Y‐27632 (10 μM, 30 min before ouabain) and caspase inhibitors (50 μM, 2h before ouabain); caspase‐2 inhibitor z‐VDVAD‐fmk, caspase‐3 inhibitor Z‐DEVD‐fmk and pan caspase inhibitor z‐VAD‐fmk on cell death were examined. Treatment of ouabain, digoxin and paclitaxel resulted in concentration dependent cell‐substratum detachment and cell death. Neither Y‐27632 nor caspase inhibitors did not prevent drug‐induced cell detachment. Ouabain, digoxin and also paclitaxel treatment induced cleavage of Rock II in both HeLa and HepG2 cells. Although this cleavage was not inhibited by Y‐27632, all the caspase inhibitors prevented the proteolytic clevage of Rock II. It has been demonstrated that activation of caspase‐3 results in cleavage and activation of caspase‐2. Therefore our results point out that drug treatment induces activation of caspase‐3, which produces active caspase‐2 causing proteolytic cleavage of Rock II in cancer cells during cell death. As a conclusion, cleavage of Rock II by caspase‐2 is a common process in a cell‐type independent manner in cell death.

This study is supported by TUBITAK in 110S426 project.
Potential Inflammatory Effects of Levodopa – Insights Into Parkinson's Disease Therapy
Authors: Allen Erin MG , Mieyal John J
Published: June 2021

Inflammation and oxidative stress are implicated in the onset and progression of Parkinson's disease (PD), a debilitating movement disorder of the elderly. Remarkably, the drug of choice for symptomatic treatment of PD, L‐DOPA, is a pro‐oxidant found to be cytotoxic to SH‐SY5Y cells (a common neuronal model), and it exhibits adverse clinical effects. The goal of this project is to investigate the influence of the neuronal environment on L‐DOPA neurotoxicity. Elevated levels of TNFα, a pro‐inflammatory cytokine, are found in PD patients and their mononuclear cells treated with L‐DOPA. Our hypothesis is that L‐DOPA stimulates an inflammatory response in microglia and astrocytes that exacerbates neurotoxicity. We found that L‐DOPA promoted the release of TNFα from BV‐2 cells (immortalized microglia) under serum‐free conditions. The net neurotoxic effect of L‐DOPA in SH‐SY5Y cells co‐cultured with BV‐2s was also monitored, however the study was limited by the growth conditions of SH‐SY5Y cells. Primary astrocytes and microglia were found to release inflammatory cytokines upon L‐DOPA treatment, yet were insensitive to L‐DOPA‐induced toxicity. Collectively, these data suggest an inflammatory response to L‐DOPA treatment that may contribute to the progressive constellation of symptoms of PD. Supported in part by a VA merit review grant (JJM, BX000290) and NIH training grant (EMGA, T32DK007319).
PKC phosphorylation of Med25 is critical for Mediator assembly: Implications for HNF4α signaling in drug metabolism
Authors: Zhang Shu‐Yun , Surapureddi Sailesh , Goldstein Joyce A.
Published: June 2021

HNF 4α, a member of the nuclear receptor superfamily regulates a variety of critical metabolic pathways with the help of conserved multisubunit Mediator complex that acts as a functional interface between regulatory transcription factors and the general RNA polymerase II initiation apparatus. Med25, a variable partner of Mediator complex has been found to enhance RNA polymerase II recruitment significantly and stabilize transcription complexes at the promoter of drug metabolism genes. Our lab has previously shown that Med25 plays a pivotal role in modulating HNF4α; transcription activity, there by regulating drug metabolism in primary human hepatocytes. Here, we report that Med25 is phosphorylated by PKC subunits as determined by in vitro phosphorylation assays, inhibition of PKC subunits, and mutation of the phosphorylation sites. We postulate that the phosphorylation status of Med25 is critical for dephosphorylation of HNF4α; and phosphorylation of RNA Pol –II (CTD). Our findings suggest that Med25 phosphorylation by PKC is a novel regulatory mechanism that promotes association with Mediator complex and, as such, may facilitate a feed‐forward action of HNF4α; signaling in drug metabolism.
Impaired endothelium‐dependent vasorelaxation contributes to hypertension in leukocyte‐specific protein 1 (LSP1)‐deficient mice
Authors: Liu Lixin , Jiang Bo
Published: June 2021

LSP1 is an intracellular, Ca2+‐and F‐actin‐binding protein expressed in leukocytes and endothelial cells. Adult male LSP1‐deficient (Lsp1−/−) mice had higher blood pressure than their WT counterparts. By Western blot, LSP1 was found present in aorta and mesenteric artery tissues in WT mice but not in Lsp1−/− mice. The role of LSP1 in regulating arterial vascular tone was studied. The contraction of aorta and mesenteric arteries in response to phenylephrine and angiotensin II was similar in WT and Lsp1−/− mice. The relaxation of aorta artery was more sensitive to sodium nitroprusside and methacholine in Lsp1−/− mice than in WT mice. The phenylephrine‐constricted mesenteric arteries showed identical relaxation curve to sodium nitroprusside in these two strains of mice, but had no relaxation to methacholine in Lsp1−/− mice or in denuded WT mesenteric arteries. Methacholine‐induced mesenteric arterial relaxation in WT mice was suppressed partially by the pretreatment of L‐NAME or the combination of apamin + TRAM‐34, abolished completely by the combination of L‐NAME + apamin + TRAM‐34, but not changed by indomethacin. Our results demonstrate the defects in endothelium‐dependent vasorelaxation in resistance arteries in mice deficient in LSP1 revealing an important role for LSP1 in endothelium‐dependent vasodilation in small resistance arteries and blood pressure regulation.
Overexpression of stem cell niche factor fibroblast growth factor‐2 transgene in the renal medulla attenuated sodium retention in Dahl S rats
Authors: ZHU QING , Hu Junping , Han Wei‐Qing , Li Pin‐Lan , Li Ningjun
Published: June 2021

Adult stem cells play important roles in maintaining normal organ functions. Fibroblast growth factor‐2 (FGF2) is a fundamental stem cell niche factor that stimulates the proliferation and differentiation of stem cells. We showed before that there were defects in FGF2 and stem cells in the renal medulla in Dahl S (DS) rats, suggesting that the impairment of FGF2 and consequent stem cell deficiency may contribute to the renal medullary dysfunction in DS rats. The present study determined whether improvement of stem cell population by overexpression of FGF2 transgene in the renal medulla would recover the renal medullary dysfunction and attenuate the Na+ retention in DS rats. FGF2 overexpression plasmid was transfected into the renal medulla in uninephrectomized DS rats for 10 days. FGF2 transfection significantly increased stem cell marker CD133+ cell numbers (0.89±0.29% in FGF2 rats vs. 0.21±0.02% in control) in the renal medulla and enhanced urinary Na+ excretion by 16% compared with control after acute IV Na+ loading. Furthermore, chronic high salt‐induced Na+ retention was remarkably attenuated in FGF2‐treated rats (10.55± 2.86 mmoles/day vs. 18.56± 1 in control). It is suggested that correction of the impairment in FGF2‐mediated regulation of stem cell function in the renal medulla improves the renal medullary dysfunction in DS rats. (support: NIH grants HL89563, HL106042 and DK54927).
Central cardiovascular function during a cold challenge in raynaud's patients
Authors: Ball Guy , Hussain Zahra , Davis John E
Published: June 2021

The purpose of this study was to examine the relationship between the ischemia seen during a Raynaud's attack and the central cardiovascular response. It was hypothesized that during cold conditions (12° C ice bath for the hand) Raynaud's patients would demonstrate lower cardiac output (CO), coinciding with ischemia in the hand. Ten female subjects (20.2±1.0 years), five with Raynaud's (RAY) and five control (CON), completed two trials: a baseline visit and a cold‐induced visit where the hand was placed in a cold container at 12° C. Cardiac output, mean arterial pressure (MAP) and heart rate (HR) were measured at rest, during occlusion (at 250 mmHG), and after occlusion. CO was measured using a CO2 re‐breathing technique (Collier). CO, HR, and MAP were measured at each of the three times. As expected, resting CO, HR, and MAP were not different between groups (CO 3.9 ±.11 L/min, HR 73.0±9.6 bpm, and MAP 87.4±1.4 mmHG for CON; CO 3.8±.38 L/min, HR 75.6±12.3 bpm, and MAP 87.1±2.3 mmHg for RAY). Contrary to the original hypothesis however, RAY demonstrated a higher CO during the cold test than CON (4.3± .20 L/min vs. 3.6± .10 L/min, P<0.05). This disparity was greatest during post‐occlusion readings (CON 3.4 ± 1.0 vs. RAY 4.5± .9 L/min). These findings suggest a response by Raynaud's patients in which increasing cardiac output and blood flow will ultimately provide a correction for the regulatory error in the ischemic hand.
Measurement of cardiac function using dual catheterization in the Dahl SS rat on low salt
Authors: Sanders Matthew D. , Tewari Shivendra , Kurth Terry , Ryan Robert P. , Cowley Allen W. , Beard Daniel A. , Carlson Brian E.
Published: June 2021

The assessment of cardiovascular function in both animal and human studies has traditionally focused on left ventricular pressure and volume measurements. However, quantifying the interaction of left ventricular function with the right ventricle and cardiovascular system as a whole extends this functional assessment to the systems level. Computational models of the heart and circulatory system incorporating left and right ventricular function have been developed but experimental data describing simultaneous function of both right and left side of the heart have not been previously measured. Here, we studied cardiac performance in rats by measuring left ventricular pressure‐volume and right ventricular pressure simultaneously using a pressure‐volume conductance catheter in the left ventricle and a pressure‐tip catheter in the right ventricle. This study focuses on development of the experimental protocol and using it to assess normal cardiovascular function in the Dahl SS rat (SS/JrHsdMcwiCrl) on low salt as a baseline for comparative studies. It was found that this dual catheterization measurement method sufficiently informs our computational model, defines cardiac and cardiovascular system function and provides an excellent platform for future comparison of cardiovascular system function between strains on different diets such as Dahl SS and SS.13BN on low and high salt.
Lenti‐Angiotensin‐(1–7) transduction of Islet+ cardiac progenitor cells improves the reparative capacity in Doxorubicin induced Cardiomyopathy
Authors: Bruce Erin , Qi Yanfei , Horowitz Alana , Raizada Mohan K. , Katovich Michael J.
Published: June 2021

Doxorubicin (DOX), a highly effective antineoplastic agent, can have devastating effects on the heart. Recent studies indicate that cardiac progenitor cell (CPC) loss plays a major role in the pathophysiology of DOX‐induced cardiomyopathy. We have recently demonstrated the potential for Angiotensin‐(1–7) (Ang‐(1–7)) to improve the beneficial effects of CPCs when given as therapy in a model of myocardial infarction. We hypothesize that giving CPCs transduced with lenti‐Ang‐(1–7) will further improve the regenerative capabilities of CPC therapy in DOX‐induced cardiomyopathy. Male SD rats were injected via tail vein with 2.5mg/kg/week DOX for 5 weeks, reaching a cumulative dose of 12.5mg/kg. 4 × 10^8 Islet+ CPCs, or lenti‐Ang‐(1–7) CPCs, were injected via jugular vein 3 days post Dox injection at weeks 3 and 5. Hemodynamic parameters were measured 2 weeks later. DOX significantly reduced dP/dtmax (3769 ± 322 vs 6880 ± 345 for control, mmHg/sec) and ventricular mass (VM, 1.75 ± 0.09 vs 3.07 ± 0.2 for control, (g/cm)). In comparison to DOX, CPCs alone restored dP/dtmax by 7.5% and increased VM by 5.3%. Ang‐(1–7) expressing CPCs further improved dP/dtmax by 13.8%, and increased VM by 8.3%. Ang‐(1–7) transduced CPCs seems to further improve the reparative capacity of CPC therapy and may prove to be a significantly beneficial therapy in preventing DOX‐induced cardiomyopathy.