bluuebunny/crossref_metadata_2025 huggingface dataset:
Blockade of T‐type calcium channel with R(−) efonidipine ameliorates the renal ischemia induced by angiotensin II in Sprague‐Dawley rats
Authors: Mori Takefumi , Hu Chunyan , Guo Qi , Yoneki Yoshimi , Nakamichi Takashi , Ito Sadayoshi
Published: June 2021

Blockade of T‐type calcium channel (TCC) has been shown to protect from renal injury. We have previously demonstrated ‐that blockade of TCC with R(−) efonidipine (R(−)EFO), a specific TCC blocker, could selectively increase renal medullary blood flow (MBF) in Sprague‐Dawley (SD) rats. Present study was designed to determine whether blockade of TCC with R(−)EFO would ameliorate the renal medullary ischemia induced by angiotensin II (AngII) in SD rats. Renal cortical blood flow (CBF) and MBF were simultaneously monitored by laser‐Doppler flowmetry in anesthetized SD rats. Following 30 min. baseline, AngII was intravenously infused for 30 min (50 ng/kg/min), and then R(−)EFO (0.25mg/h, intravenously) was infused together for 90 min and compared to the vehicle group. Urine was collected for hydrogen peroxide excretion (UH2O2V) measurement. Infusion of AngII significantly reduced renal CBF and MBF by 22±3.4% and 28±1.7% (P<0.05), respectively. However, the reduction of MBF was significantly attenuated by R(−)EFO (P<0.05). No significant change was observed in CBF between the two groups. Moreover, R(−)EFO significantly attenuated the increase in UH2O2V induced by AngII (P<0.05). We conclude that blockade of TCC with R(−)EFO would attenuate AngII‐induced reductions in renal MBF by altering AngII‐induced renal oxidative stress. Supported by the Grants‐in‐Aid from the Min. of ECSST of Japan (No. 17590181)
Expression of dynamin and nitric oxide synthase (NOS) isoforms in rat and mouse collecting ducts
Authors: Hyndman Kelly Anne , Kim Eujin , Pollock Jennifer S
Published: June 2021

Dynamins are a family of proteins (DNM1, DNM2 and DNM3) involved in facilitating endocytosis. Recently, a report linked increased dynamin activity to S‐nitrosylation of DNM. We reported that DNM2 and DNM3 interact with NOS1 and NOS3 in the rat kidney inner medulla. The renal inner medulla is comprised of various tubular and vascular structures. The purpose of this study was to determine the expression of NOS and DNM isoforms in isolated rat and mouse renal collecting ducts (CD) and whether DNM is S‐nitrosylated in the CD. Primary suspensions of CDs were prepared by collagenase digestion from rat and mouse inner medulla. CD suspensions and mIMCD3 cells were homogenized and Western blotting analysis for NOS and DNM isoforms performed. Rat CD suspensions express NOS1α, NOS1β, and NOS3; whereas mouse CD suspensions and mIMCD3 express NOS1β and NOS3. Rat CD, mouse CD, and mIMCD3 cells express DNM2 and DNM3. We found little to no expression of NOS2 or DNM1 in any of the CD preparations. Rat CDs were immunoprecipitated with anti‐DNM2 followed with immunoblotting with anti‐S‐nitrosocysteine. We found that DNM2 in rat CD is S‐nitrosylated. In conclusion, these three different models can be used to test questions of DNM/NOS interactions in the CD. This work was funded by NIH HL60653 to JSP; NKF Postdoctoral fellowship to KAH; MCG Diabetes and Obesity Discovery Institute Scholar award to EK.
Ion translocation via the transmembrane domain 5 in the sodium/bicarbonate transporter NBCe1
Authors: LEE SOOJUNG , CHOI INYEONG
Published: June 2021

We previously demonstrated that the charge‐conserved substitution of Asp at position 555 with a Glu in NBCe1 (D555E) alters the anion selectivity of the transporter. This mutant protein normally produces HCO3− currents, but can elicit Cl− currents in CO2/HCO3−‐free solution. Thus, we hypothesized that Cl− binds to the mutant transporter at the HCO3− binding site if HCO3− is not present. To test the hypothesis, we expressed D555E in Xenopus oocytes and assessed Cl− currents and HCO3− currents using two‐electrode voltage clamp. Under Cl−‐free conditions, the mutant transporter produced an outward current upon exposure to Cl− (104 ± 28 nA, n = 5). The Cl− current was markedly reduced in CO2/HCO3− solution (28 ± 5, n = 5). However, the Cl− current was smaller than Cl− currents in Na+‐free CO2/HCO3− (48 ± 20, n = 5). Current‐voltage relationships showed that the slope in CO2/HCO3− approached to the slope in Na+‐free CO2/HCO3− (p < 0.05, n = 6 for each), indicating the current in Na+‐free CO2/HCO3− is mediated by Cl−. Replacing transmembrane domain 5 (TM5) of NBCe1 with the corresponding TM5 of the electroneutral Na/HCO3 transporter NBCn1 produced outwardly rectifying currents that are progressively smaller at positive voltages, where electrogenic Na/HCO3 transport should be favorable. We conclude that TM5 of NBCe1 containing Asp555 is responsible for electrogenicity of the transporter.
Role of pH regulatory components in the transduction response of central CO2 chemoreceptors
Authors: Cordovez Juan M , Clausen Chris , Moore Leon C , Solomon Irene C
Published: June 2021

Increased levels of CO2 (hypercapnic acidosis, HA) acidify central CO2 chemoreceptors resulting in a change in neuronal excitability that drives ventilation. Recently, we developed a mathematical model that combines a minimal Hodgkin‐Huxley (HH)‐based expression of neuronal excitability with a robust transport model based on the Goldman‐Hodgkin‐Katz (GHK) equation, and used this model to study excitability in response to HA. We found that HA induced small passive changes in ionic composition that modulated membrane potential (Vm), but the firing rate changes observed were not wholly consistent to those reported in the literature, suggesting that pH‐sensitive currents may be required. Although previous HH‐based single‐compartment neuron models have been developed to investigate CO2 chemoreception, these models contain only an estimate of pH regulatory fluxes. To overcome the limitations of the existing models, we have (1) expanded our model to include pH‐sensitive currents proposed to participate in CO2 chemoreception and (2) identified the role of each current in modulation of Vm. We found that the expanded model with kinetic descriptions for pH regulatory mechanisms, HA‐induced small passive changes in ionic composition, and pH‐sensitive currents best captures the neuronal response to HA. We suggest that all components be included in transduction models of CO2 chemoreception. Supported by NS045321
Modulation of neuroventilation and central chemosensitivity: serotonergic and hypocretinergic effects
Authors: Corcoran Andrea E , Richerson George B , Harris Michael B
Published: June 2021

Hypocretin (HC; orexin) neurons modulate arousal state and possibly breathing. The HC system regulates dorsal raphe serotonin (5HT) neurons. Potential for HC modulation of medullary raphe 5HT neurons, putative central chemoreceptors, is unknown. We show HC receptors 1 and 2 (HC‐r1, HC‐r2) on rat medullary raphe 5HT neurons, and potential for HC modulation of chemosensitive medullary 5HT neurons. In an in situ perfused juvenile rat preparation, HC‐1 and SB‐408124 (HC‐r1 antagonist) does not alter baseline phrenic nerve firing (neuroventilation), while blocking HC‐r1 prevents hypercapnic neuroventilatory responses (chemosensitivity). Thus, endogenous HC modulation is necessary, but not sufficient, for chemosensitivity. Exogenous HC‐1 again does not alter hypoglossal neuroventilation in a neonatal rat in vitro medullary slice, or slices derived from neonatal wildtype and knockout mice (Lmx1b) which lack all central 5‐HT neurons. Lmx1b slices do not respond to acidosis, confirming that 5‐HT neurons are critical for central chemosensitivity.HC facilitate baseline neuroventilation in part through 5‐HT neurons. The impact of HC on 5‐HT neuron sensitivity to acidosis remains in question. NIH 2U54NS041069‐06A1
Effects of bilateral inhibition of retrotrapezoid nucleus on breathing in conscious rats
Authors: Takakura Ana C , Moreira Thiago S , de Paula Patricia M , Menani Jose V , Colombari Eduardo
Published: June 2021

The last five years many studies suggested the retrotrapezoid nucleus (RTN) as the central respiratory chemoreceptors, however all experiments were performed in anesthetized animals. In the present study we sought to investigate a possible role of RTN neurons in the normal breathing in conscious rats. Male Holtzman rats (280–300 g) with bilateral stainless steel cannulas implanted into the RTN were used. In conscious rats, bilateral inhibition of RTN neurons with the GABA‐A agonist muscimol (100 pmol/50 nl) reduced resting ventilation (120±62, vs. saline: 928±77 ml/min/kg) and the increase in ventilation produced by hyperoxic hypercapnia (10% CO2) (962±174, vs. saline: 2654±231 ml/min/kg). However, muscimol into the RTN did not change the increase in ventilation produced by activation of peripheral chemoreceptors with hypoxia (8% O2) (1294±116, vs. saline: 1552±108 ml/min/kg). Bilateral injections of muscimol into the RTN slightly reduced resting MAP (Δ = −13±7, vs. saline: +3±2 mmHg), but did not affect the changes in MAP and HR produced by hyperoxia hypercapnia and peripheral chemoreceptor stimulation. In conclusion, RTN neurons activated by pCO2 presumably via their intrinsic chemosensitivity, provide chemical drive to breathe during resting or hypercapnia.

Financial support: FAPESP and CNPq.
Gamma Linolenic Acid Levels in Atherogenic Dieted Rabbits with Primrose Oil Supplementation
Authors: Beard Elizabeth Letitia , Rodriguez Gustavo A.
Published: June 2021

The effect of primrose oil (PO), containing 9% gamma linolenic acid (GLA), 74.6% linoleic Acid and 9% oleic acid, addition to an atherogenic diet (AD) fed to New Zealand strain, male rabbits, on the fatty acid contents of samples of their aortae, kidney and lung tissues, was studied. Five Control animals (ADC Group) were fed an Atherogenic Diet (AD) containing 92% Purina Rabbit Chow, 2% cholesterol and 6% corn oil. Five Experimental rabbits (APO Group) were fed AD with 5% PO added. After 10 weeks of maintenance on these diets, the animals were sacrificed by nembutal injection. One gram tissue samples from lung, aorta and kidney were analyzed by gas chromatographic assay for their fatty acid content. Palmitic Acid was significantly higher in APO Lung samples: 38.4. vs 29 mol% in the ADC group. Cis Linoleic Acid levels were markedly higher in the APO Aortae: 53.4 vs 40.2% mol % in ADC tissues. APO Kidney assays found 39.6 vs 36.1 mol % in ADC samples. Alpha Linolenic Acid (18:3W3) levels were significantly higher in ADC Lung fractions: O.6 vs 0.06 mol% in APO samples. ADC Aortae mean values for 18:3W3 were 1.9 vs 1.13 mol % in APO tissue and in ADC Kidney samples, it measured 0.63 vs 0.38 mol% for APO tissue. Gamma Linolenic Acid (18: 3W6) levels in APO Lung were 0.82 vs 0.44 mol% in ADC samples. In APO Aortae, GLA values were 1.49 vs 0.05 mole % in ADC tissues. APO Kidney GLA values were 0.86 vs 0.245 mol% in ADC fractions. Diets that can induce increased GLA levels in tissues may allow for decreased tissue inflammation and atherogenesis by increasing the availability of the fatty acid precursors needed for the synthesis of major antiatherogenic prostaglandins.
Gene inflammatory expression profiling in right versus left ventricles in young urbanites: what is the long‐term impact of myocardial inflammation in the setting of air pollution?
Authors: Villarreal‐Calderon Rodolfo , Palacios‐Moreno Juan , Parker Keith , Calderon‐Garciduenas Lilian
Published: June 2021

Increased cardiac morbidity and mortality are associated with air pollution. Mexico City (MC) residents are exposed to high concentrations of fine particulate matter and endotoxin. South MC residents are exposed to higher endotoxin v North (Rosas 2007). SMC exposed mice had the highest CD14 and IL1beta mRNA in myocardium v N (Villarreal 2009). To test the hypothesis that residency within MC is a key factor for the degree of myocardial inflammation, we measured inflammatory mediator genes in autopsy samples from R and L ventricles of 6 South and 15 North age‐matched MC residents 18.5±5.2y. South residents had significant up‐regulation of CD14, IL1beta and TNFalpha v North (p<0.001). An anti‐inflammatory biventricular response was also present: IL10 (RV p=0.001, LV p=0.02). Increases in myocardial IL1beta and TNFalpha have been implicated in the pathogenesis of myocardial dysfunction and cardiomyocyte death in ischemia‐reperfusion injury, sepsis, chronic heart failure, etc. Exposure to polluted urban air is associated with myocardial inflammation in young people, the RV responds differently from the LV and endotoxin is a key player in the inflammatory response. Occult cardiotoxicity (Golomb et al., 2009) may be a deleterious effect in young urbanites and the long‐term impact of sustained myocardial inflammation is uncertain.
The role of CXCR3 signaling in myocardial infarction
Authors: Dobaczewski Marcin Piotr , Bujak Marcin , Gonzalez‐Quesada Carlos , Xia Ying , Li Na , Lu Bao , Frangogiannis Nikolaos Georgios
Published: June 2021

Chemokine signaling regulates the reparative response following myocardial infarction and plays an important role in the pathogenesis of cardiac remodeling. CXCR3, the main functional receptor for IP‐10, Mig and I‐TAC, is markedly induced in healing myocardial infarcts. We have recently demonstrated that IP‐10 critically regulates infarct healing by inhibiting fibroblast migration. Our current investigation tests the hypothesis that IP‐10 actions in the infarcted myocardium may be mediated through CXCR3. Accordingly, we examined the effects of CXCR3 loss on infarct healing and cardiac remodeling. The CXCR3 ligands IP‐10 and MIG but not I‐TAC were upregulated in reperfused mouse infarcts. Flow cytometry analysis of cell suspensions harvested from mouse infarcts after 72h of reperfusion identified large populations of CD3+ T lymphocytes, CD14+ monocytes, CD31+ endothelial cells and alpha‐smooth muscle actin +/collagen I+ myofibroblasts expressing CXCR3. Much like IP‐10 −/− mice, CXCR3 −/− mice exhibited increased infiltration of the infarcted myocardium with neutrophils and macrophages. However, in contrast to the increased infiltration of the infarct border zone with myofibroblasts observed in IP‐10 −/− mice, CXCR3 −/− hearts had reduced and delayed myofibroblast infiltration. In vitro experiments demonstrated that the inhibitory effects of IP‐10 on bFGF ‐induced cardiac fibroblast migration were independent of CXCR3. In contrast to the increased remodeling observed in IP‐10 deficient mice, CXCR3 null mice and WT animals had comparable dilation following infarction. The findings suggest that the inhibitory effects of IP‐10 on fibroblast migration protect from the development of adverse remodeling and are mediated through CXCR3‐independent pathways.
Hydrogen Peroxide Upregulates PLGF and VEGF‐A Expression in Primary Human Coronary Artery Smooth Muscle Cells
Authors: Shaw Jennifer H. , Lloyd Pamela G.
Published: June 2021

When supply arteries become occluded a compensatory mechanism termed arteriogenesis occurs, resulting in collateral artery enlargement. Arteriogenesis involves attachment of monocytes to the endothelium and subsequent vessel wall infiltration. Placental growth factor (PLGF) mediates components of this collateral remodeling process. Our lab previously showed that hydrogen peroxide (H2O2) significantly upregulates PLGF mRNA expression in A10 cells, an established smooth muscle cell (SMC) line derived from rat aorta. Since SMC undergo phenotypic changes in culture, the goal of this study was to determine whether PLGF is also regulated by H2O2 in a more physiological model (primary SMC). Primary human coronary artery SMC (CASMC, Lonza) were grown to ~80% confluence, then serum‐starved (1% FBS) for 48 hrs. CASMC were then treated with exogenous H2O2 (50 μM) (N=3) for 4 hrs. PLGF and VEGF‐A gene expression was analyzed by real time quantitative PCR (Q‐PCR). PLGF gene expression was increased ~3.5 fold (p<0.05) in CASMC by H2O2 treatment, compared with untreated CASMC (N=3). VEGF gene expression increased ~6 fold (p<0.05) in response to H2O2. We conclude that H2O2 regulates PLGF and VEGF expression in primary SMC, confirming the physiological relevance of this mechanism. Further experiments to characterize protein levels of PLGF, VEGF, and PLGF/VEGF dimeric forms are underway. Support: NIH R01 HL‐084494 (PL).
Activation of Angiotensin II mediated hypertension in a rat model of Polycystic Ovary Syndrome.
Authors: Yanes Licy Lorena , Davis Deborah , Romero Damian G , Reckelhoff Jane F
Published: June 2021

Polycystic ovary syndrome (PCOS) affects 15 % of reproductive age women, and is characterized by hyperandrogenism, polycystic ovaries and amenorrhea. Cardiovascular risk factors are greatly increased in PCOS patients. The hypothesis to be tested was that activation of angiotensin II mediated hypertension in a PCOS rodent model. Dihydrotestosterone (DHT) (7.5 mg/90 days) pellets were implanted subcutaneously in female SD rats at 4 weeks of age (PCOS model) and compared to placebo. After 1 mos of DHT treatment, Enalapril (250 mg/l) was given in the drinking water for 2 mos. Mean arterial pressure (MAP) was measured by radiotelemetry the last 2 weeks of DHT treatment. In another cohort of rats the components of intrarenal and systemic renin angiotensin system were evaluated. PCOS rats had higher MAP compared to placebo (96±1 vs. 108±2 mm Hg p 0.05). Enalapril treatment abolished the increase in MAP in the PCOS model (108±2 vs. 77±2 mmHg p 0.01). Plasma renin activity was suppressed in the PCOS model (35.33 ±6.67 vs. 17.63 AI/ng/24hs). In contrast, intrarenal angiotensinogen and ACE mRNA levels were significantly up regulated in the model. Intrarenal AT1R mRNA and protein level were significantly down regulated in the PCOS rats. In summary, activation of intrarenal renin angiotensin system plays a major role in the pathophysiology of hypertension in PCOS model.
High muscle triglyceride content in obesity is not due to elevated lipogenic enzyme activity or to the suppression of lipolytic proteins
Authors: Li Minghua , Paran Christopher , Wolins Nathan E , Horowitz Jeffrey F
Published: June 2021

We compared key factors that regulate intramyocellular triglyceride (IMTG) metabolism and IMTG trafficking between obese (OB) and lean (LN) women. Skeletal muscle biopsy samples from 8 OB and 9 LN women (BMI: 38±1 and 23±1kg/m2) were obtained after an overnight fast. Despite a nearly 2‐fold greater IMTG concentration in OB vs LN (75±15 vs 40±8 umol/g dw; p<0.05), we found protein abundance of the IMTG synthetic enzyme diacylglycerol acyltransferase 1 (DGAT1) to be lower in OB (p<0.01), with a similar trend for a lower DGAT activity in OB (370±47 vs 441±57 pmol/min/mg; p=0.18). Neither the protein level nor activity of the glycerol‐3‐phosphate acyltransferase (GPAT) was different between groups. We found no differences between groups in muscle adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) protein abundance, and no differences in phosphorylation of specific sites known to affect HSL activity. Protein abundances of the IMTG trafficking proteins perilipin 2 and 3 were lower (p<0.05) in muscle from OB vs LN when expressed relative to IMTG content. Our findings indicate that the elevated IMTG content found in obese women was not due to an up‐regulation of key lipogenic proteins, or to the suppression of lipolytic proteins. The impact of lower levels of IMTG trafficking perilipins remains to be clarified.

Supported by NIH Grant #R01 DK071955
Oxidative stress enhances myofibrillar protein degradation via calpain and caspase‐3
Authors: Smuder Ashley J , Kavazis Andreas N , Hudson Matthew B , Nelson W Bradley , Powers Scott K
Published: June 2021

Oxidative stress has been shown to accelerate proteolysis and muscle fiber atrophy during periods of prolonged skeletal muscle inactivity. Currently, the mechanisms connecting oxidative stress to muscle protein degradation remain unknown. In this regard, calpain and caspase‐3 are proteases capable of degrading select myofilament proteins in skeletal muscle. However, it is unclear if oxidation of myofibrillar proteins increases recognition and degradation by these key proteases. Therefore, we tested the hypothesis that oxidative modification of myofibrillar proteins increases their susceptibility to degradation by calpain and caspase‐3. Myofibrillar proteins were isolated from skeletal muscle and exposed to in vitro oxidation to produce varying levels of protein modification. Modified proteins were then independently incubated with active calpain I, calpain II, or caspase‐3 and protein degradation was assessed via peptide mapping. Our results reveal that increased protein oxidation results in a stepwise escalation in the degradation of myofibrillar proteins by calpain I, calpain II, and caspase‐3. These findings provide a link to connect oxidative stress with accelerated proteolysis during disuse muscle atrophy.

Supported by NIH RO1 HL087839 awarded to SKP
Candesartan reverses depression‐like behavior in a rodent model of depression
Authors: Stedenfeld Kristen A , Clinton Sarah M , Kerman Ilan A , Akil Huda , Watson Stanley J , Sved Alan F
Published: June 2021

Depression and cardiovascular disease (CVD) share a bidirectional relationship wherein the presence of one increases the likelihood of the other. Antagonists of the renin‐angiotensin system (RAS) that are useful in treating CVD may also have antidepressant properties. Therefore, the current study examined the impact of chronic treatment with an AT1 receptor antagonist on behavioral and cardiovascular parameters in a rodent model of depression. Rats selectively bred for low locomotion in a novel environment have been shown previously to be particularly vulnerable to develop depression‐like behavior in response to chronic mild intermittent variable stress (CMS). Rats were exposed to 4 weeks of CMS and anhedonic behavior was assessed via weekly sucrose preference tests (SPT). Heart rate (HR) and HR variability (HRV), a clinically relevant measure of autonomic function, were measured by radiotelemetry. Previously demonstrated effects of CMS (reduced SPT scores, increased HR, decreased HRV) were observed. Candesartan (0.5mg/kg/day, sc infusion) or vehicle was administered and CMS was continued. In CMS‐exposed rats, candesartan markedly improved SPT scores and reduced measures of anxious behavior. These results suggest that the RAS might be a useful therapeutic target for the treatment of co‐morbid depression and CVD. [N000140210879, NIDA 5P01DA021633 (HA) 1K99MH081927, NARSAD Young Investigator (IAK)]
Investigation of Insulin Signaling in Parkinson's Disease Cytoplasmic Hybrid Cells
Authors: Morris Jill Kathleen , Esteves A. Raquel , Bomhoff Gregory L. , Swerdlow Russell H. , Stanford John A. , Geiger Paige C.
Published: June 2021

Neurodegenerative diseases, including Parkinson's disease (PD), have been linked to Type 2 Diabetes (T2D), and impaired mitochondrial function occurs in both diseases. A cell model of PD, the PD cytoplasmic hybrid (cybrid), contains mitochondrial DNA from PD patients. Control (CTL) cybrids contain mitochondrial DNA from healthy individuals. PD cybrids exhibit increased stress kinase activity (JNK and IkKB) compared with CTL cybrids. Because stress kinases are linked to insulin resistance, we hypothesized that PD cybrids would exhibit deficits in insulin signaling. The aim of this study was twofold: to characterize differences in insulin signaling between PD and CTL cybrids and to investigate the impact of increased heat shock protein (HSP) expression on insulin signaling. Insulin resistance occurs due to post‐receptor serine phosphorylation of the insulin receptor substrate (IRS) proteins. Compared to CTL cybrid lines, PD cybrids exhibited increased serine phosphorylation of IRS2, the primary IRS in the brain, and increased JNK activation. In addition, heat treatment resulted in HSP70 upregulation and a concomitant decrease in IRS2 serine phosphorylation in PD cybrids. HSPs can inhibit the action of stress kinases, which is a potential mechanism for decreased IRS2 serine phosphorylation. Upregulation of HSPs may be a valuable intervention in preventing insulin resistance in PD.
Stress‐induced variation in protein utilization and plasma glucose in a male passerine bird from urban and rural populations
Authors: Rodriguez Natalie , Davies Scott , Deviche Pierre , Sweazea Karen
Published: June 2021

In mammals, stress increases the secretion of glucocorticoids, resulting in the mobilization of energy substrates such as glucose. We investigated the mobilization of glucose and proteins in a wild‐caught male passerine, the Abert's Towhee, Papilo aberti, in response to mild acute stress. Birds were captured from urban and rural populations and blood samples were collected immediately after capture and 60 minutes later (after handling and mild restraint‐induced acute stress). Birds were then transported to the laboratory where they were allowed to acclimate for 12–38 days prior to collecting another blood sample. Plasma glucose (PGlu) did not increase in response to acute stress, but significantly increased by 11.8% and 5.5% in urban and rural birds, respectively, after birds were brought into captivity. This increase may be related to the ad libitum availability of high quality food. No further changes in PGlu were observed over the following 38 days. Plasma uric acid significantly decreased by 34.1% in birds from urban locations and 51.7% in birds from rural locations in response to acute stress, suggesting decreased protein utilization in both populations. Neither PGlu nor plasma uric acid differed between birds originating from urban and rural populations. Ongoing studies are aimed at measuring plasma glycogen to determine if glycogenolysis contributes to the captivity‐associated increase in PGlu.
Thioamide derivative modulates annexin v expression in human liver carcinoma (HepG2) cells
Authors: Jackson Shannon A. , Yedjou Clement , Cameron Joseph A. , Tchounwou Paul
Published: June 2021

Thioamide is one of a group of antithyroid drugs prescribed in the treatment of hyperthyroidism. Thioamide derivative is currently used for the treatment of thyroid disease, tuberculosis, and leprosy. Recent in vitro experiment in our laboratory demonstrated thioamide is highly cytotoxic to human liver carcinoma (HepG2) cells. Although published studies indicate that thioamide derivative has medicinal properties effective against many diseases other than liver cancer, the molecular mechanisms under which this compound induces apoptosis of cancer cells remain largely unknown. In the present study, we proposed to use human liver carcinoma (HepG2) cells as a test model to evaluate the basis apoptotic mechanism of thioamide derivative. To achieve this goal, the expression of annexin V was measured by flow cytometric analysis. Human liver carcinoma (HepG2) cells were treated with different doses of thioamide derivative for 48 hours. Results from the flow cytometric assessment (Annexin V FITC/PI) showed a strong dose‐response relationship between thioamide derivative exposure and annexin V positive cells undergoing early stage apoptosis in HepG2 cells. Taken together, our results indicate that phosphatidylserine externalization was involved in thioamide derivative induced toxicity to HepG2 cells. This grant is supported in part by NIH‐NIGMS Grant R25GM50117 and in part by NIH Grant 2G12RR013459‐11.
Acute Alcohol Intoxication Inhibits Stem Cell Antigen‐1 Expression by Gr1+ Myeloid Lineage Committed Precursor Cells during Bacteremia
Authors: Melvan J. Nicholas , Siggins Robert W. , Zhang Ping , Nelson Steve , Bagby Gregory
Published: June 2021

Hematopoietic precursor cells preferentially commit toward the granulocyte lineage during bacterial infection. Our previous studies have shown that alcohol intoxication impairs stem cell antigen‐1 (Sca1) expression by lineage negative hematopoietic precursor cells during bacterial infection, a key component of the granulopoietic response. It remains unknown whether alcohol affects the Sca1 response in myeloid lineage committed (Gr1+) cells. Thirty minutes after intraperitoneal injection of alcohol (5g EtOH/kg), male Balb/c mice received an intravenous injection of E. coli and BrdU. Bacteremia increased the number of Gr1lo and Gr1hi cells expressing Sca1. Increased Sca1 expression during bactermia was accompanied by greater BrdU incorporation and CFU activity in Gr1lo cells compared to controls. In culture, 12hrs of LPS exposure stimulated the expression of Sca1 by sorted Gr1+Sca1‐ cells. Alcohol intoxication suppressed the Sca1 response, BrdU incorporation, and CFU activity of Gr1lo cells. These data show that alcohol suppresses Sca1 expression in cells committed to myeloid lineage development during bacteremia. Inhibiting the Sca1 response in downstream myeloid cells may serve as a mechanism of alcohol induced myelosuppression and thereby impair host defense against bacterial infection. NIH NRSA AA09803, AA017494, and AA07577.
Valproic acid enhances Oct4 promoter activity in myogenic cells
Authors: Teng Han Fang , Kuo Yu‐Liang , Loo Moo Rung , Li Chung Leung , Chu Ta Wei , Suo Hsien , Liu Hang Seng , Chen Shen Liang
Published: June 2021

Induced pluripotent stem cells (iPS) are derived from somatic cells through ectopic expression of stem cell‐specific transcription factors, including Oct4, Nanog, Sox2, Lin28, Klf4 and c‐Myc. Although these iPS cells are similar to embryonic stem (ES) cells in their pluripotency, a few defects, such as insertion mutagenesis, employment of oncogenes, and low efficiency, associated with the procedure of creating them have hindered their clinical application. A study has shown that concomitant treatment with valproic acid (VPA) can significantly enhance the efficiency and avoid the usage of oncogenes. To elucidate the mechanisms of VPA enhanced pluripotency, we stably transfected a luciferase reporter (Oct4‐1.9k‐Luc) driven by the promoter of Oct4 into P19 embryonic carcinoma (EC) cells and C2C12 myoblasts to examine their response to VPA. By treating them with retinoic acid (RA) and VPA, we found that VPA could both activate Oct4 promoter and rescue its inhibition by retinoic acid. VPA treatment also enhanced endogeneous Oct4 expression but repressed that of MyoD in C2C12 myoblasts. These observations suggest that VPA enhances pluripotency by direct activating Oct4 transcription and repressing those of differentiation genes. Besides, our results imply that VPA may exert its effect through specific promoter‐targeting factors instead of through general effects on chromatin structure.
Role of Epo in angiogenesis, development, maintenance of muscle mice fibres in normoxia and hypoxia
Authors: LAUNAY Thierry , Hagstrom Luciana , Agbulut Onnik , El Hasnaoui‐Saadi Raja , Marchant Dominique , Richalet Jean‐Paul , Beaudry Michèle
Published: June 2021

Erythropoietin (Epo) and vascular growth factor (VEGF) are known to be involved in the regulation of cellular activity when oxygen transport is reduced like in anemia or hypoxic conditions. Because it has been suggested that Epo could have a role in skeletal muscle development, repair and angiogenesis we aimed to assess Epo‐deficiency using Epo‐deficient trangenic (Epo‐TAgh) mouse model in both normoxia and hypoxia. By histoimmunology, ELISA and real time PCR we did not observe any muscle fibre atrophy or accumulation of active HIF1‐α but an improvement of microvessel network and an up‐regulation of VEGFR2 mRNA in Epo‐deficent gastrocnemius compare to wild type one. In hypoxia both models exhibit an up‐regulation of VEGF120 and VEGFR2 mRNA but no accumulation of Epo protein. EpoR mRNA is not up‐regulated in both Epo‐deficient and hypoxic gastrocnemius. In conclusion muscle deconditioning observed in patients suffering from renal failure are not due to Epo‐deficency.
Cellular signals associated with integrin activation in afferent arteriole
Authors: Yip Kay‐Pong Daniel , Lo Chun‐Min , Sham James S.K.
Published: June 2021

By pulling fibronectin‐coated magnetic beads to activate integrins in renal vascular smooth muscle cells (VSMCs) grown on flexible substratum, it was found that the traction force exerted on the substratum by VSMC remained elevated after the termination of pulling force. These observations showed that activation of α5β1 integrins (receptors for fibronectin in VSMCs) with mechanical force triggered myogenic‐like behavior in VSMCs. We sought to determine whether antibodies known to activate α5β1 integrins can elicit cellular signals related to myogenic response in VSMCs of perfused afferent arterioles. We monitored VSMC [Ca2+]i with fluo‐4 and ROS (reactive oxygen species) production with CM‐H2DCFD using confocal microscopy. ROS has been implicated in signaling events of myogenic response in other vascular beds. α5‐integrin antibody ( HMα5‐1, 10 μg/ml) induced an initial increase of [Ca2+]i ( 124 ± 8 % of baseline, 35 cells/5 vessels), which was followed by multiple Ca2+ spikes. The increase of [Ca2+]i was associated with reduction of luminal diameter. α5‐integrin antibody also increased ROS production, which reached the peak ( 110 ± 3% of baseline, 7 vessels) at about 30 s after the application. Control antibodies did not trigger Ca2+ mobilization and ROS production. Our observations suggested Ca2+ mobilization is the major response when α5β1 integrins were activated in renal VSMCs. Supported by AHA.
Exogenous L‐Arginine (L‐Arg) Reverses Angiotensin II (AngII)‐Mediated Renal Cortical and Medullary Vasoconstriction and Improves Pressure Diuresis and Natriuresis
Authors: Das Satarupa , Mattson David L
Published: June 2021

We previously demonstrated that exogenous L‐Arg restores the blunted pressure diuresis and natriuresis relationship and normalizes arterial blood pressure in AngII‐infused rats. The present study determined the role of L‐Arg to modulate total renal blood flow and renal cortical and medullary blood flow in anesthetized, AngII‐infused, Sprague‐Dawley rats (n>6/group). Renal blood flow was measured by ultrasonic flowmetry and regional renal blood flow was evaluated with the use of implanted optical fibers for laser‐Doppler flowmetry. Aortic clamps were utilized to maintain renal perfusion pressure at 110 mmHg throughout the experiments. Intravenous infusion of AngII (20 ng/kg/min) resulted in a significant decrease in total renal blood flow from 7.2 ± 0.5 ml/min/gkwt to 5.3 ± 0.3 ml/min/gkwt. The AngII infusion did not significantly alter superficial cortical blood flow but significantly decreased medullary blood flow by 19 ± 4%. Co‐infusion of L‐Arg (300 μg/kg/min) reversed the effects of AngII to decrease renal blood flow and renal medullary perfusion. The present data demonstrate that AngII‐mediated vasoconstriction of the renal cortical and medullary vasculature is reversed by exogenous L‐Arg; these experiments indicate an important role for nitric oxide to blunt the renal vasoconstrictor, anti‐diuretic, and pro‐hypertensive effects of AngII. Supported by HL29587 and DK062803.
Evidence for Protein Kinase C Dependent Stimulation of Reactive Oxygen Species in Isolated Nuclei of Renal Epithelial Cells
Authors: Wilson Bryan Anthony , Gwathmey TanYa , Pirro Nancy , Chappell Mark C
Published: June 2021

We previously reported evidence for functional angiotensin (Ang) receptors in isolated nuclei of the rat kidney. Ang II stimulates AT1 receptors on nuclei to increase the formation of reactive oxygen species (ROS) through a protein kinase C (PKC) pathway. To identify a potential cell model for this intracellular system, we examined ROS production and PKC isoform expression in the NRK52E cell line derived from rat renal proximal tubules. Nuclei were isolated from confluent cells by hypoosmotic shock, differential centrifugation and subsequent separation through 0.8M sucrose. To measure ROS, nuclei were loaded with the dichlorofluorescein (DCF, 10 μM) in the absence or presence of either the H2O2 scavenger ebselen (10 μM), an NADPH oxidase inhibitor DPI (10 μM) and the PKC inhibitor Bisindolylmaleimide I (PKCi, 10 μM). Nuclei were then exposed to the PKC agonist phorbol myristate acetate (PMA, 1 μM) for 10 min and the fluorescence measured in a plate reader. PMA significantly increased ROS production (79 + 21%, n=5) above the control nuclei. The PMA stimulation was essentially abolished by both ebselen (14 + 7%) and the PKCi (−2.0 ± 25%) but partially inhibited by DPI [46 ± 13%]. Immunoblots of nuclei revealed both PKC‐α; and PKC‐δ; isoforms. In summary, we demonstrate that isolated nuclei from NRK52E cells contain a functional PKC system that is coupled to the formation of ROS. Funding: HL‐56973, HL‐51952, R25GM064249
Motor Unit Recruitment Order in Diaphragm Muscle Following Spinal Cord Injury
Authors: Seven Yasin Baris , Mantilla Carlos B. , Zhan Wen‐Zhi , Sieck Gary C.
Published: June 2021

Motor units of the diaphragm muscle (DIAm) are recruited in an orderly fashion across ventilatory and non‐ventilatory tasks. Spinal hemisection at C2 (C2HS) blocks descending ipsilateral input to phrenic motoneurons and causes DIAm paralysis. With time after C2HS, there is spontaneous recovery of phrenic nerve activity ipsilateral to the injury. We hypothesized that recruitment order of DIAm motor units is maintained following recovery of DIAm electromyographic (EMG) activity post‐C2HS. DIAm EMG activity in adult male rats was recorded chronically using a pair of electrode wires implanted into the midcostal region bilaterally. DIAm EMG activity was strongly correlated to transdiaphragmatic pressure (Pdi) measured using esophageal and gastric balloons across different motor behaviors (Eupnea, hypoxia‐hypercapnia (10%O2 and 5%CO2), tracheal occlusion, fictive sneezing and bilateral supramaximal phrenic nerve stimulation). Fictive sneezing produced forces comparable to those elicited by phrenic nerve stimulation, and thus DIAm EMG activity during fictive sneezing was considered maximal. Absence of DIAm EMG activity was verified at the time of C2HS and 3 days (3D) post‐C2HS. Over time, a subset of animals displayed functional recovery of rhythmic DIAm EMG activity ipsilateral to C2HS, although DIAm EMG activity was reduced when compared to pre‐injury levels. EMG activity increased across ventilatory and non‐ventilatory tasks associated with increasing drive in C2HS animals displaying functional recovery, supporting a generalized strengthening of contralateral presynaptic drive over time following spinal cord injury.
Mathematical Characterization of the Inhibitory Effect of Mg2+ on the Kinetics of Mitochondrial Ca2+ Uniporter
Authors: Pradhan Ranjan K. , Qi Feng , Beard Daniel A. , Dash Ranjan K.
Published: June 2021

Ca2+ is a key regulatory ion and changes in mitochondrial Ca2+ homeostasis can lead to cellular dysfunction and apoptosis. Ca2+ is transported into respiring mitochondria via Ca2+ uniporter, which is inhibited by Mg2+. In spite of a large number of experimental studies, the kinetic mechanism associated with the Ca2+ transport and kinetic mechanism of Mg2+ inhibition of the uniporter function is not clear. To gain a quantitative understanding of the effect of Mg2+ on mitochondrial Ca2+ regulation, we developed a biophysically‐based kinetic model of Ca2+ uniporter on the basis of its kinetic properties and a presumed mechanism of Mg2+ inhibition of the uniporter function. The model is extended from our previous model of Ca2+ uniporter (Dash et al, Biophys J 96: 1318–1332, 2009) which is based on a multi‐state catalytic binding mechanism for carrier‐mediated transport and Eyring's free energy barrier theory for transformation. The model satisfactorily describes the available data on the kinetics of Ca2+ uniporter and appropriately depicts the inhibitory effect of Mg2+ on the uniporter function, in which Ca2+ uptake is hyperbolic in the absence of Mg2+ and sigmoid in the presence of Mg2+. This model can be useful for developing mechanistic models of mitochondrial bioenergetics and Ca2+ handling to understand the mechanisms by which Ca2+ plays a role in mediating signaling pathways and modulating energy metabolism.
N‐methyl D‐aspartate (NMDA) receptors endow dendrites of gonadotropin releasing‐hormone (GnRH) neurons with additional active properties
Authors: Suter Kelly , O'Boyle Michael , Hemond Peter
Published: June 2021

Dendrites of GnRH neurons express tetrodotoxin‐sensitive voltage‐gated sodium channels and generate action potentials. Other dendrites exhibiting action potentials possess additional active properties such as NMDA‐mediated potentials. We tested the hypothesis that GnRH dendrites express R1 subunits of NMDA receptors and exhibit amplification of sub‐threshold, excitatory post‐synaptic potentials (EPSPs) via NMDA receptor activation. Immunochemistry indicated GnRH dendrites express R1 subunits. Stimulating living GnRH dendrites with simulated sub‐threshold EPSPs resulted in plateau potentials and individual action potentials. When GABAergic inputs were blocked with picrotoxin (100 μM), repetitive action potentials occurred. The NMDA receptor antagonist D‐2‐amino‐5‐phosphono‐valeric (50 μM) eliminated responses. In small diameter dendrites, NMDA receptors would have a pronounced impact. Activating simulated NMDA conductances (at 560 μm dendrite length) in our compartmental models of GnRH neurons resulted in repetitive action potentials. Activating proximal NMDA receptors (at 200 and 100 μm dendrite length) diminished the impact of NMDA receptors. Our findings indicate GnRH dendrites exhibit regionalized expression of the NMDA conductance thereby forming location‐dependent microdomains where estrogen‐sensitive GABAergic neurons can regulate local excitability.
Some biological consequences of climate change
Authors: St James John , Buckeridge Stewart
Published: June 2021

Uniformitarianism permits understanding of the past on the basis of the present, and modelling the future through consideration of the fossil record. This paper addresses the impact climatic change has had on acorn barnacles and eucalyptus trees. Acorn barnacles (Balanomorpha) are first recorded after the K/T mass‐extinction event. In the Palaeogene, rapid radiation resulted in their occupying most marine environments. That balanomorphs survived both the Palaeocene‐Eocene thermal maximum and the Pleistocene glaciation is testament to their ability to adapt to opportunities; they are known from the littoral (Chthamalus) to 3600m (Tetrachaelasma) and within this from diverse substrates: rock, wood and miscellaneous flotsam, plus in symbiosis or commensalism with most marine organisms. Darwin's (1854) view of the late Tertiary as the “age of barnacles” is reflected in their diversity, distribution and biomass. Barnacles are contrasted with the Australian Myrtaceae: plants ranging from woody shrubs to tall trees. The most significant is Eucalyptus s.l. which typifies Australia's flora, and is characterised by aromatic leaves which produce eucalyptol. Eucalyptus has evolved strategies that result in its domination of Australian open woodlands – these include production of highly flammable eucalyptol oil (with a flashpoint of 49°C) and an unprecedented ability to regenerate following forest fires.
β‐Catenin mediates TGF‐β1‐induced myofibroblast differentiation in a matrix stiffness‐dependent manner: implication to aortic valve sclerosis
Authors: Chen Jan‐Hung , Chen Wen Li Kelly , Sider Krista , Simmons Craig
Published: June 2021

Introduction

In aortic valve sclerosis, myofibroblast differentiation and activation of the TGF‐ß1 and ß‐catenin pathways are observed in the fibrosa, the stiffer layer of the leaflet.

Objectives

To investigate the roles of ß‐catenin and matrix stiffness in TGF‐ß1‐induced myofibroblast differentiation.

Methods

Sclerotic aortic valves were from pigs fed an atherogenic diet. Porcine aortic valve interstitial cells (VICs) were studied in vitro on collagen‐coated polyacrylamide gels with tunable stiffness.

Results

In sclerotic valves, myofibroblasts colocalized with TGF‐ß1, Wnt3a, ß‐catenin, and pSmad2/3 in the fibrosa, which was significantly stiffer than the ventricularis (P=0.006). TGF‐ß1 induced pSmad2/3‐dependent ß‐catenin nuclear translocation in VICs more readily on matrices with fibrosa‐like stiffness (P<0.05). Degrading ß‐catenin pharmacologically inhibited TGF‐ß1‐induced myofibroblast differentiation (P=0.04) without altering pSmad2/3 activity. Conversely, increasing ß‐catenin stability with Wnt3a alone did not induce differentiation (P=0.73). However, combining TGF‐ß1 and Wnt3a caused greater myofibroblast differentiation than TGF‐ß1 treatment alone (P=0.004). These novel results suggest that myofibroblast differentiation in valve sclerosis involves matrix stiffness‐dependent cross‐talk between TGF‐ß1 and Wnt signaling pathways.

Support: HSFO, NSERC.
Function of Angiostatin in Acute Lung Inflammation
Authors: Aulakh Gurpreet Kaur , Liu Lixin , Suri Sarabjeet Singh , Singh Baljit
Published: June 2021

ALI is characterized by the migration of activated neutrophils into lungs and causes significant mortality. Although necessary, activated neutrophils also cause significant tissue damage. The mechanisms of neutrophils migration and pathogenesis of ALI are not fully understood. Angiostatin binds to ATP synthase and integrins, and inhibits angiogenesis. We investigated effect of subcutaneous administration of angiostatin in a mouse model of E. coli lipopolysaccharide (LPS) induced ALI. Immunohistochemistry showed higher expression of angiostatin/plasminogen in the airways and blood vessels in the inflamed lungs. Angiostatin expression was increased in inflamed lung extracts as well as bronchoalveolar lavage (BAL) supernatant (P<0.007). Angiostatin treatment of the LPS‐treated mice decreased total cell counts and protein concentration (P<0.05) but increased (P<0.05) apoptotic neutrophils in BAL and decreased (P<0.05) myeloperoxide in lung extracts. The protein and mRNA expression of cytokines and chemokines (IL‐1β, KC, MCP‐1 and MIP‐1α) was not altered in LPS+angiostatin mice. We conclude that angiostatin expression is increased in inflamed lungs and angiostatin treatment inhibits neutrophil migration into the inflamed lungs and lung inflammation. (Funding NSERC Discovery Grant)

Grant Funding Source: NSERC Discovery Grant
The pathophysiology of vascular calcification in a rodent model of chronic kidney disease
Authors: Shobeiri Navid , Beseau David , Phelan Rachel , Holden Rachel M. , Adams Michael A.
Published: June 2021

Vascular calcification (VC) occurs in aging populations, but is markedly accelerated in patients with chronic kidney disease (CKD). Through manipulation of dietary adenine, we produced a Sprague Dawley rat model characterized by graded severity of CKD with VC. Circulatory changes were determined by arterial radiotelemetry (n=6) and by assessment of aortic pulse wave velocity (PWV; n=32). VC was assessed using the calcium‐O‐cresophthalein assay and von Kossa staining. At sacrifice, kidney function (creatinine (μmol/L)) was worst in the CKD group with VC (251±60 μmol/L), compared to CKD alone (200±69 μmol/L) or control (50±16 μmol/L). Longitudinal serum phosphate measures revealed that those CKD animals with the highest serum phosphate mid‐study and prior to the development of circulatory changes (i.e. radiotelemetry) subsequently developed calcification (5.96±1.3 mM in calcifying CKD versus 4.4±1.3 mM in CKD versus 2.8±0.2 mM in control). VC animals also demonstrated elevated PWV (3.2±0.3 log(cm/s) vs control 2.9±0.1 log(cm/s)) and widening pulse pressure ((PP) 38±4.7 mmHg to 58 ±15.2 mmHg). Furthermore, there were positive correlations between the phosphate regulating hormone, fibroblast growth factor 23, and PP with left ventricular hypertrophy (LVH; r2=0.32 & r2=0.58 respectively, p<0.005). Hyperphosphatemia and VC in CKD are likely mechanistically linked and are key factors in enhanced LVH.