bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of the renal sympathetic nerves in a model of postmenopausal hypertension
Authors: Maranon Rodrigo Oscar , Lima Roberta , Juncos Luis A. , Reckelhoff Jane F
Published: June 2021

The mechanisms responsible for the increase in blood pressure (BP) in postmenopausal women are unknown. Our preliminary studies suggest that the sympathetic nervous system may contribute to the hypertension (HT) in aging female SHR. The aim of this study was to determine whether the renal nerves contribute to HT in old and young female SHR. Female SHR (18 and 3 mos of age, respectively, n=4/grp) underwent uninephrectomy, and two weeks later, underwent unilateral renal denervation (O‐RD and Y‐RD) or sham surgery (O‐S and Y‐S) and telemetry transmitter implantation. After two weeks recovery, mean arterial pressure (MAP) and heart rate (HR) were recorded for 5 days. MAP was higher in O‐S than other groups (O‐S:167.5±2.4 vs O‐RD:138±2.7 mmHg; p<0.001; and Y‐S:140.6±6.3 vs Y‐RD:122.1±3.9 mmHg, p<0.001). The reduction in MAP after denervation was greater in O‐RD (O‐RD:29.5±0.5 mmHg, vs Y‐RD:17.0±0.9 mmHg, p<0.001). Renal NE content was significantly decreased compared to shams (O‐S: 201.1±30.3 pg/mg vs O‐RD: 35.6±10.3 pg/mg wet weight, p<0.001; and Y‐S:239±24.5 pg/mg vs Y‐RD:18.1±11.4 pg/mg wet weight, p<0.001). These data indicate that the renal nerves play a key role in the control of BP in pre‐ and post‐cycling SHR. The data also suggest that the renal nerves contribute to HT more in old females than young.
Sea water acidification alters cell volume regulation and discharge in jellyfish
Authors: Marino Angela , Morabito Rossana , La Spada Giuseppina , Lauf Peter K , Adragna Norma C.
Published: June 2021

Increased acidification/PCO2 of sea water is a threat to the environment and affects marine animals’ homeostasis. In this study, the effect of sea water pH changes was assessed on the stinging cells, nematocytes, of jellyfish (Cnidarians’ Scyphozoan) collected in the Strait of Messina, Italy, which possess venom containing nematocysts. Two features were studied: 1) regulatory volume decrease (RVD), a homeostatic parameter common to all cells, in nematocytes, and 2) nematocyst discharge with release of toxins, used for Cnidarians’ defense and aggression strategies. To test the effect of pH on RVD, nematocytes were isolated with 605mM NaSCN and subjected to 35% hyposmotic stress in artificial sea water (ASW), at normal (7.65) or acid pH (4.5). Untreated nematocytes, after their expected swelling, regulated their volume within 15 min by activating K+ and Cl‐channels and cotransporters, whereas RVD was abrogated at pH 4.5, indicating a pH‐dependence. Nematocyst discharge was assessed in acid ASW (pH 4.5) plus 1% trypsin. Under these conditions, and in contrast to normal pH, no discharge response was observed. In conclusion, these data indicate that Cnidarians’ homeostasis and function may be altered by environmental changes, such as sea water acidification, validating jellyfish as a bioindicator for marine environmental quality monitoring.
Na+,HCO3−‐cotransport is crucial for intracellular pH control in human breast cancer
Authors: Boedtkjer Ebbe , Moreira José , Mele Marco , Vahl Pernille , Jespersen Marie , Christiansen Peer , Jensen Vibeke , Pedersen Stine F. , Aalkjaer Christian
Published: June 2021

Single nucleotide polymorphisms in Na+,HCO3−‐cotransporter NBCn1 (SLC4A7) are associated with breast cancer. Here, we studied the expression of NBCn1 and the importance of Na+,HCO3− ‐cotransport in human and mouse breast carcinomas. NBCn1 expression was detected at mRNA‐ and protein level in freshly obtained human breast tumors. Immunohistochemical membrane staining for NBCn1 was increased in human primary breast carcinomas and metastases compared to matched healthy breast epithelium. Using pH‐sensitive fluorophores, we found that Na+,HCO3 −‐cotransport and Na+/H+‐exchange are the only important mechanisms of net acid extrusion from human and ErbB2‐overexpressing mouse primary breast carcinomas. In the near‐physiological pHi range (pHi>6.60), CO2/HCO3−‐dependent mechanisms were responsible for >90% of total net acid extrusion. Steady‐state pHi of human breast carcinomas was 0.35±0.06 units lower in the absence than in the presence of CO2/HCO3−. In conclusion, NBCn1 expression is increased in human breast carcinomas where Na+,HCO3−‐cotransport is a prominent mechanism of acid extrusion and crucial for steady‐state pHi control. Support: Danish Council for Independent Research and Danish Cancer Society.
Mathematical modeling of the role of carbonic anhydrase II and IV on the influx of CO2 in a Xenopus oocyte
Authors: Occhipinti Rossana , Musa-Aziz Raif , Boron Walter F.
Published: June 2021

Exposing oocytes to a solution containing CO2/HCO3 causes the familiar fall in intracellular pH (pHi) and a rise in surface pH (pHS) followed by a decay. Musa‐Aziz et al (ASN, 2005) examined the effects of carbonic anhydrases (CAs) on pH transients caused by CO2 influx. They found that injecting CAII into oocytes or expressing CAIV on the oocyte surface accelerates the initial rate of pHi acidification (dpHi/dt) and amplifies the height of the pHS spike (ΔpHS). They proposed that both enzymes enhance CO2 influx by maximizing CO2 gradients across the plasma membrane. We use a mathematical model of a Xenopus oocyte—which accounts for CO2, carbonic acid (H2CO3), HCO3, protons, and a multitude of non‐HCO3 buffer pairs—to investigate the above findings. We simulate the experimental protocol in which the CO2/HCO3 solution is delivered from the bulk to the oocyte surface by raising exponentially the concentrations of CO2, H2CO3 and HCO3 in the bulk and assuming that initially no CO2, H2CO3 and HCO3 are present in the extracellular unconvected fluid. We use the experimental data for 1.5% CO2/10mM HCO3 (pHo=7.5) to find parameter values that simultaneously match ΔpHS, the time that it takes for pHS to reach its peak (tP), dpHi/dt, and the time delay in the pHi decay. We validate the model against data collected when exposing oocytes to 5% CO2/33mM HCO3 and to 10% CO2/66mM HCO3. The model confirms that CAII and CAIV enhance CO2 influx.
Chelerythrine, a protein kinase C inhibitor, abolishes K flux through Na/K pump and Na‐K‐2Cl cotransporter‐1, and stimulates SK channels in human lens epithelial cells
Authors: Lauf Peter K , Lepera Michael A , Adragna Norma C
Published: June 2021

Chelerythrine (CET) is a widely used selective protein kinase C (PKC) inhibitor and proapoptotic agent. Lens cataract formation has been associated with apoptosis and cellular K loss/Na gain. The effect of short‐term CET exposure on both K loss and K uptake, using Rb as congener, was studied in human lens epithelial cells (HLECs) to characterize the role of PKCs in pro‐apoptotic membrane cation transport changes. Ouabain (O), bumetanide (B) and Cl replacement by NO3 were applied to study the Na/K pump, Na‐K‐2Cl cotransporter‐1 (NKCC1), and K‐Cl cotransporter (KCC), respectively, and cations were measured by atomic absorption/emission spectrophotometry. Exposure to 50 μM CET (20 min) inhibited the total, O‐, and OB‐sensitive Rb influxes via Na/K pump and NKCC1 by >90%, with IC50 values of ~ 30 and ~20 μM CET, respectively, without significant KCC activity change. K loss (20–30%) at 20–30 μM CET was 4‐aminopyridine‐insensitive but inhibited by apamin. Since ATP levels did not change, the Na/K pump inhibition may be due to loss of a PKC‐mediated phosphorylation, or direct drug/transporter interaction. Loss of NKCC1 function confirms reported role of PKCδ, present in HLECs, whereas intermediate CET concentrations opens PKC‐regulated SK channels. Apparently, CET affected independently all three transport functions.
Store‐operated calcium channel Orai and STIM regulated by high glucose
Authors: DASKOULIDOU NIKOLETA , Jiang Hongni , Zeng Bo , Atkin Stephen L , Xu Shang-Zhong
Published: June 2021

The Orai and Stim genes are recently identified as store‐operated calcium channel molecules, which play important roles in essential physiological and pathophysiological functions from gene expression to cell growth. Oxidative stress‐induced endothelial dysfunction is the major etiology for several common diseases, such as hyperglycemia and diabetes. Here we aimed to investigate the expression and function of Orai and Stim and their regulation by high glucose in vascular endothelial cells.

The mRNA and protein expression of Orai1–3, STIM1 and STIM2 were detected by real‐time PCR and western blotting in human endothelial cell line EA.hy926 and human blood vessels. Orais and STIMs were upregulated by the chronic treatment with 25 mM high glucose (HG) for 60 hours. The store‐operated Ca2+ influx was enhanced after chronic treatment with HG, however, no effect was observed in the acute experiment with HG perfusion. HG showed no direct effect on Orai1–3 channel activity detected by patch clamp and STIM1 translocation monitored by imaging system in the HEK‐293 cells co‐expressing with Orai and Stim1 genes. The endothelial cell migration and angiogenesis were impaired after HG treatment for 72 hours.

Our results suggest the importance of store‐operated channel molecules in the pathophysiology of diabetic blood vessels. This work was supported in part by British Heart Foundation and The Leverhulme Trust.
A mutation in a CLC anion channel alters serotonergic neuronal activity in C. elegans
Authors: Branicky Robyn , Miyazaki Hiroaki , Strange Kevin , Schafer William
Published: June 2021

Egg‐laying in C. elegans is controlled by the HSN neurons, which release serotonin to activate egg‐laying muscles. The dominant egl‐42(n995) mutation prevents serotonin release and inhibits egg‐laying. Intracellular Ca2+ imaging demonstrated that HSN activity is reduced in egl‐42 mutants. Whole genome sequencing revealed that egl‐42(n995) is a point mutation in the clh‐3 gene, which encodes the CLC anion channel splice variants CLH‐3a and CLH‐3b. Overexpression of both wild‐type and mutant clh‐3 phenocopies the egl‐42(n995) egg‐laying defect, while clh‐3 deletion leads to hyperactive egg‐laying. The n995 allele is a valine to alanine mutation that is located in membrane helix P, which contributes to the monomer‐monomer interface in CLC homodimers. This residue is highly conserved in all human CLC proteins except CLC‐Ka and CLC‐Kb. CLH‐3a and CLH‐3b mutant channels exhibit increased current amplitude, a strongly depolarized activation voltage, and more rapid voltage dependent activation kinetics. CLH‐3a and CLH‐3b mutants also exhibit loss of pre‐potentiation behavior and greatly reduced sensitivity to inhibition by GCK‐3 kinase, respectively. Our results demonstrate 1) that CLC helix P plays an important role in controlling channel voltage sensitivity and 2) that clh‐3 encoded channels likely regulate HSN membrane potential and hence Ca2+ entry and neuronal excitability.
Association of Epithelial Na+ channel (ENaC) and Acid‐Sensitive Ion Channel (ASIC) Subunits with Lipid Rafts in High Grade Gliomas
Authors: Chebukati Janetricks Nanjala , McNicholas Carmel M , Fuller Catherine M
Published: June 2021

Malignant gliomas, the most common type of primary tumors of the central nervous system, are divided into four grades based on their degree of malignancy. Glioblastoma multiforme (GBM), Grade IV, is the most frequently occurring, most invasive, and has the worst prognostic outcome. Studies in our laboratory have established that high grade glioma cells express subunits of the Epithelial Na+ channel/Degenerin family, and that these cells also exhibit a basally active cation current which is not present in low grade tumor cells or normal astrocytes. The goal of our research is to characterize the pathways that regulate trafficking of this novel channel complex in gliomas by testing the hypothesis that this heteromeric channel complex is recruited to and assembled on lipid rafts prior to incorporation into the plasma membrane. By utilizing biochemical and biophysical methods, we have obtained data showing an association of the lipid raft markers caveolin‐1 and flottilin‐1 with ENaC and ASIC subunits. Cholesterol depletion experiments indicate decreased expression of these subunits in lipid raft fractions isolated from glioma cells. Overall, the results generated from these studies will provide important clues as to the physiological role of this current pathway in the biology of brain tumors. Research supported by NIH‐5T32DK7545‐23.
Annexin A2 acts as scaffolding receptor for plasminogen and tissue plasminogen activator in renal epithelia
Authors: Kaminski Henriette Josephine , Dathe Christin , Neymeyer Hanna , Brand Daniel , Loof Tanja , Peters Harm , Bachmann Sebastian , Paliege Alexander
Published: June 2021

Annexin A2 has been shown to act as a receptor for plasminogen (Plg) and its tissue type activator (tPA) in endothelial cells (EC). Aim of the present study was to elucidate the renal distribution of the components of this complex under control conditions and in a Thy1 nephritis model of proteinuric renal disease. Thy1 nephritis was induced by intravenous injection of OX7 anti‐Thy1 antibody (1mg/kg body weight). Distribution of Plg, tPA and ANXA2 was studied by immunofluorescence in controls and after 5 or 15 days of OX7 injection. Immunoreactive structures were further characterized by double‐labeling immunofluorescence using antibodies against rat endothelial cell antigen for EC, Na(+),K(+),2Cl(−) cotransporter for thick ascending limbs (TAL), aquaporin‐1 for thin descending limbs (tDL) and aquaporin‐2 for collecting ducts (CD). As expected, all three proteins were identified in EC of controls and Thy1 animals. In addition we found overlapping signal for tPA, Plg and ANXA2 in the basolateral and apical membrane of tDL, TAL and CD both, in treated and untreated animals. Furthermore, abundance of tPA and Plg in the apical membrane of TAL and CD was increased in proteinuric animals. In conclusion, our results provide evidence for the presence of a proteolytic complex in the apical membrane of renal epithelia where it may play a protective role in the setting of proteinuric disease.

support: DFG FOR 667
Visualization of exocytosis and vesicle trafficking in inner medullary collecting duct (IMCD)
Authors: Yip Kay-Pong , Amin Jahanshah , Cha Byeong
Published: June 2021

Conventional confocal microscopy has been used to monitor vasopressin (AVP) induced apical exocytosis in perfused IMCD using FM1‐43 as a membrane tracer, but individual fusion event could not be resolved. In two‐photon confocal microscopy, two‐photon excitation does not excite tracer molecules outside of the focal plane, and it can simultaneously excite multiple tracers with a single laser source. We sough to determine whether two‐photon confocal microscopy can detect individual vesicle fusion event in IMCD using a membrane tracer (FM1‐43) and a fluid‐phase tracer (Sulforhodamine B, SRB) simultaneously. Luminal FM1‐43 and SRB were excited at 850 nm. AVP (1 nM) induced a gradual increase of FM1‐43 fluorescence in the apical membrane, indicating an increase of membrane surface area due to exocytosis. Opening and flattening of fusion pores were also observed in the apical membrane. At the site of membrane fusion, FM1‐43 emission was increased ahead of SRB emission, which suggested that FM1‐43 diffuses along the lipid membrane into the vesicle while SRB enters the vesicle after the fusion pore is opened. The SRB emission dropped after the vesicle was flattened. The size of vesicles varied between 0.5 to 1.5 μm. The fusion time ranged from 30–80 s. Transcellular trafficking of fluorescent vesicles was also observed. These results demonstrated the feasibility to monitor vesicle fusion and trafficking in perfused IMCD.
Modulation of urinary bladder paracellular permeability by stretch
Authors: Carattino Marcelo D , Prakasam Herman S , Ruiz Wily G , Apodaca Gerard
Published: June 2021

In response to bladder filling and voiding, umbrella cells undergo changes in cell‐shape and vesicular trafficking. However, it is not known how filling/voiding impact paracellular transport in these cells. To investigate how changes in umbrella cell stretch affect paracellular permeability, bladder mucosa was mounted in Ussing chambers and studied under current clamp conditions. In this system the transepithelial resistance (TER) was dependent on whether the tissue was bowed outwards, simulating filling, or bowed inwards as observed during voiding when the epithelium is refolded. Outward bowing provoked by an increase in mucosal hydrostatic pressure of ~1 cm of water caused a decrease in junctional resistance from 21.1 ± 8.5 to 1.8 ± 0.4 kΩ.cm2. These effects were reversible and dependent on the applied force. The integrity of the tight junctions (TJ) was assessed by examining the distribution of ruthenium red (RR) added to the mucosal surface of the tissue and examined by electron microscopy. The TJ appeared intact and no RR was found along the basolateral surface of the cells during experimental filling. Finally, depletion of extracellular Ca2+ by EGTA completely disrupted the TER of control, but not of stretched uroepithelium, indicating a change in the junctional barrier. In summary, our results indicate that filling and voiding impacts the junctional complex of umbrella cells.
Decreased expression of adipose fatty acid transporters CD36 and FATP1 contributes to increased plasma free fatty acids during prolonged fasting in northern elephant seal pups
Authors: Viscarra Jose Abraham , Rodriguez Ruben , Vazquez-Medina Jose Pablo , Champagne Cory D. , Adams Sean H , Crocker Daniel E , Ortiz Rudy M.
Published: June 2021

The northern elephant seal endures a 2–3 month fast during which it depends primarily on the oxidation of fatty acids to meet its energetic demands. Plasma free fatty acid (FFA) concentration increases and is associated with the onset of insulin resistance in late‐fasted pups. Plasma FFA concentrations respond differentially to an intravenous glucose tolerance test (ivGTT) depending on fasting duration suggesting that the effects of glucose on lipid metabolism are altered. Elucidation of the mechanisms responsible for the increase in plasma FFA in late‐fasted seals is lacking. To assess the impact of fasting and glucose on lipid metabolism, adipose and plasma samples were collected before and after GTTs, performed on early (2 weeks, n=5) and late (6–8 weeks; n=8) fasted pups. Plasma triglycerides and FFAs increased post‐GTT in fasted seals. Fasting decreased adipose lipase activity by 50% and the expressions of CD36, FAS, FATP1 and PEPCK‐C 22 ‐ 43% in adipose. Fasting increased plasma lipase activity 2‐ fold. These results present the probable cause of, and reason for the onset of fasting‐induced insulin resistance in adapted mammals, as fasting leads to the altered regulation of lipolysis and reduction in tissue fatty acid uptake in late‐fasted pups, likely to ensure a constant supply of lipids in plasma, while conserving glucose for tissues which cannot metabolize lipids and sparing lean tissue. Funded by NIH HL91767
COMPETING ENERGY DEMANDS IN THE HAWKMOTH MANDUCA SEXTA
Authors: Contreras Heidy Lorena , Davidowitz Goggy
Published: June 2021

In this study we used the hawkmoth, Manduca sexta, to investigate the effects that feeding status and activity level had on the metabolic rates of insects. Overall our interest lie in understanding how these organisms prioritizes and resolves the demand for energy from two simultaneous competing demands (digestion and activity). To determine the effect of feeding on the metabolic rate of M. sexta, we measured resting metabolic rates in unfed and fed individuals. Twenty moths (10 male and 10 female) were fed one of 5 treatments (water, 5, 15, 25, 35% sucrose) at random, and their metabolic rate was measured within an hour after feeding. The energetic cost of flight for unfed and fed individuals was measured by placing individual moths in a flight mill while a flow‐through respirometry system sampled air from the mill on a second‐by‐second basis. We found that the metabolic cost of activity and digestion are independent of each other and that there is an additive effect of digesting to the metabolic cost of activity. Furthermore, we found a significant increase in the cost of processing nectar containing 15% sucrose and above, which was observed when both resting and flight metabolic rates were measured. This is the first study addressing how insects resolve the conflict between competing energy demands.
Ammonia and cortisol are transcriptional regulators of the ammonia transporter Rhcg2 in trout gill pavement cells
Authors: Nawata Michele , Wood Chris
Published: June 2021

Ammonia is the primary nitrogenous waste excreted across the gills of fish, but the mechanisms involved have never been clearly defined. Several very recent studies have now revealed that Rh glycoproteins have a role as ammonia transporters in fish gills. Based on gene expression studies, Rhcg2 appears to have a main role in this process in the pavement cells of the freshwater trout gill. Little is known however, about how these Rh genes are regulated. Using cultured trout pavement cells we have examined some of the possible transcriptional regulatory mechanisms of Rhcg2. We show that ammonia alone increases the level of Rhcg2 transcripts in the pavement cells while cortisol and 11‐deoxycorticosterone have a potentiating effect on this transcriptional response. We focus on cAMP and mitogen‐activated protein kinase pathways as potential signaling pathways involved in the regulation of Rhcg2 (NSERC Discovery).
The effect of prolonged hypoxic exposure on chemoreceptor morphology of bowfin (Amia calva) with and without access to air
Authors: Porteus Cosima S , Wright Pat A , Milsom William K
Published: June 2021

The fish literature is peppered with evidence of two types of putative oxygen chemoreceptors: internal receptors that sense changes in blood PO2 and external receptors that sense changes in water PO2. The exact location and morphology of these cells remains unknown. Our goal was to identify these elusive chemoreceptors using a facultative air breather, the bowfin. One group of fish were exposed to prolonged hypoxia (42 torr for 7 days) and had access to the surface of the water to breathe (hypoxic but not hypoxaemic). Another group of fish were exposed to the same hypoxic protocol but did not have access to the surface to breathe (hypoxic and hypoxaemic). Following exposure the fish were sacrificed and their gills sampled. We identified 3 types of putative oxygen chemoreceptors. Type I cells were found near the efferent filament artery, had no cellular projections and were immunopositive for serotonin (5‐HT), acetylcholine (ACh), synaptic vesicle (SV2) markers and were innervated (zn‐12). Type II cells were bipolar neurons found in the central venous sinus (cvs) and immunopositive for 5‐HT, ACh, SV2 and zn‐12. Type III cells were also found in the cvs, had no cellular projections and were immunopositive for 5‐HT but not for SV2 or ACh and were rarely innervated. Type I cells were significantly larger in bowfin without access to air than in bowfin with access to air, suggesting that these cells are internal chemoreceptors.
Neural Responses to Looming Objects in the Dragonfly
Authors: Gaffin-Cahn Elon , Olberg Robert
Published: June 2021

Dragonflies have high visual acuity, which, when combined with a remarkably fast visual response, allows them to hunt with a high success rate. They do so by intercepting small flies in flight rather than by chasing from behind. Eight bilateral pairs of large Target‐ Selective Descending Neurons (TSDNs) of the dragonfly ventral nerve cord respond to small, contrasting objects, which presumably represent potential prey. These interneurons are part of the neuronal circuitry that triggers small changes in wing angle and position to control flight during prey interception. In flight, dragonflies extend their legs to catch the prey about 20 ms before contact. The current research investigates the role of the TSDNs in prey contact. Spiking traces from the nerve cord were recorded during the presentation of growing black circles projected on a screen, which simulate approaching the prey. Several loom sizes and speeds were used to cover a range of realistic and unrealistic rates of expansion. We investigated whether the interneurons predict the time to contact of the simulated looming stimuli. We hypothesize that TSDNs innervate circuitry that controls leg muscles for the purpose of catching prey in flight.
Gender and age influence the ventilatory response to hypercapnia in chickens
Authors: Espinha Livia P , Macari Marcos , Bicego Kenia C , Gargaglioni Luciane H.
Published: June 2021

CO2 is a powerful respiratory stimulus for birds, however most studies do not consider the influence of gender and age in the hypercapnic ventilatory response. We evaluated blood gases and ventilatory response to hypercapnia (7% CO2 in air) of 10 (d10) and 21 (d21) days old broiler males (M) and females (F). Under resting conditions, ventilation (VE) was higher in d10 chickens compared to d21 ones, due to a higher tidal volume (VT), (M10 days: 631.3 ± 56.8 vs F10 days: 534 ± 56.9; M21 days: 84.4 ± 14 vs F21 days: 46 ± 5.3 mL kg−1 min−1). Difference in VE between M and F was observed only in d21. CO2 exposure increased total ventilation above control values, due to an increase in VT in all groups (M10 days: 1728.1 ± 289 vs F10 days: 1498 ± 213; M21 days: 240 ± 40.4 vs F21 days: 178.6 ± 36.2 mL kg−1 min−1). The CO2 response was accompanied by respiratory acidosis (pHa fell from ≈7.59 to ≈7.32) and hypercapnia (PaCO2 increased from ≈25.6 to ≈55 mmHg) in both M and F groups. Hypercapnic ventilatory response was more pronounced in d10 compared with d21, but difference between genders was observed in d21 only. Therefore, age and gender influence the normocapnic and hypercapnic ventilation in young broiler chickens. Financial Support: FAPESP, CNPq and INCT of Comparative Physiology
The Importance of Polycystin 1 (PC1) in Endothelial Mitochondrial Bioenergetics
Authors: Jones Thomas Joseph , Pung Yuh Fen , Adapala Ravi Kumar , Burke Jamie R , Thodeti Charles K , Chilian William M
Published: June 2021

PC1 is a mechanosensitive component of shear stress sensing with mutations linked to endothelial dysfunction. Since PC1 is integral to energy requiring functions, like mechanotransduction and growth, we hypothesized the loss of PC1 will effect mitochondrial energy metabolism. Therefore, we measured mitochondrial function in endothelial cells isolated from Wt and PC1 null (Pkd1null/null) mice. We determined the maximal oxygen consumption rate (OCR) and extracellular acidification rate (glycolysis) (ECAR) after uncoupling oxidative phosphorylation with 0.5 μmol/L of carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP). During uncoupling, both OCR and ECAR were significantly decreased in the Pkd1null/null cells indicating severe mitochondrial dysfunction (OCR: 470 ± 36 vs 792 ± 83 pmoles O2/min; ECAR: 36 ± 4 vs 77 ± 4 mpH/min; n=3, 4 to 5 wells/condition). In addition, FCCP produced a ~10‐fold reduction in Pkd1null/null bioenergetic reserve capacity (1851 ± 252 vs 18393 ± 512 pmoles O2, respectively). To delineate a mechanism of decreased bioenergetic capacity, cells were stained with dihydroethidium, a marker of oxidative stress. Pkd1null/null cells exhibited a heightened state of oxidative stress, consistent with the observed decrease in energy production and mitochondrial dysfunction. These results suggest the deletion of PC1 impairs mitochondrial and endothelial function.
In vivo cardiac 31P MRS in a mouse model of heart failure
Authors: Prompers Jeanine J , Bakermans Adrianus J , van Nierop Bastiaan J , Abdurrachim Desiree , Nicolay Klaas
Published: June 2021

Objective

To investigate myocardial energy status in a mouse model of heart failure using in vivo 31P magnetic resonance spectroscopy (MRS).

Methods

Male C57BL/6 mice underwent thoracic aortic constriction (TAC) surgery, inducing pressure‐overload cardiomyopathy, and were measured seven weeks after surgery (n = 5). Healthy wild‐type mice served as controls (n = 4). Cardiac cine 1H MR images were made for reference purposes and to quantify left ventricular (LV) function. Cardiac 31P MR spectra were measured from a ~6 mm cubic voxel enclosing the end‐diastolic LV myocardium using ECG triggered, respiratory gated 3D Image‐Selected In vivo Spectroscopy (ISIS).

Results

LV end‐diastolic volume and LV mass normalized to body weight were higher in TAC mice compared to controls (91.7 ± 19.0 versus 61.8 ± 6.0 μL and 4.4 ± 0.6 versus 3.1 ± 0.2 mg/g, P < 0.01), whereas LV ejection fraction was reduced in TAC mice (45.4 ± 20.0 versus 64.4 ± 5.2 %, P < 0.05). Myocardial phosphocreatine‐to‐ATP ratio was lower in TAC mice when compared to healthy controls (0.8±0.2 versus 1.2±0.2, P < 0.05).

Conclusion

Decreased EF in TAC mice is accompanied by decreased phosphocreatine‐to‐ATP ratio, indicating a disturbed energy homeostasis in this mouse model of heart failure. This research was funded by a VIDI grant from the Netherlands Organisation for Scientific Research (NWO).
Direct renin inhibition reduces SIRT3‐mediated cyclophilin D acetylation and mitochondrial dysfunction in post‐infarction cardiac remodeling in diabetic rats
Authors: Parodi-Rullan Rebecca , Barreto-Torres Giselle , Ruiz Louis , Casasnovas José , Javadov Sabzali
Published: June 2021

This study elucidates the possible contribution of mitochondria to the anti‐hypertrophic effects of the direct renin inhibitor aliskiren in post‐infarction heart failure (HF) complicated with diabetes. Diabetes was induced in male Sprague‐Dawley rats by a single injection of streptozotocin. After 7 days, the animals were randomly assigned to 4 groups: sham, HF, sham+aliskiren, and HF+aliskiren. HF was induced by coronary artery ligation for 4 weeks. Results demonstrated that HF reduced ejection fraction and cardiac output by 41% (P<0.01) and 42% (P<0.05), respectively, compared to sham‐operated hearts. Mitochondria isolated from post‐infarcted hearts demonstrated a 27% and 38% (both P<0.05) less state 3 and respiratory control index, respectively. In addition, cytochrome c loss from mitochondria was significantly higher in ligated hearts. Heart failure enhanced mitochondrial permeability transition pore (PTP) opening. In addition, HF reduced expression of the major mitochondrial sirtuin, SIRT3 with no effects on SIRT4 and SIRT5, and increased acetylation of cyclophilin D, a regulatory component of the PTP. Aliskiren significantly improved cardiac function and abrogated mitochondrial perturbations. Thus, aliskiren attenuates post‐infarction remodeling which is associated with its indirect beneficial effects on mitochondria. Supported by Novartis Pharma Corporation.
Mitochondrial Functional Specialization in Glycolytic and Oxidative Muscle Fibers: Tailoring the Organelle for Optimal Function
Authors: Burelle Yan , Hepple Russell T , Picard Martin
Published: June 2021

A unique evolutionary feature of skeletal muscle is the co‐existence within the same organs of fast (glycolytic, Type II) and slow (oxidative, Type I) muscle fibers. These fiber‐types have specific contractile properties that generate unique intracellular conditions, to which bioenergetics and mitochondrial function must be tailored. However, the extent to which mitochondrial function differs between fast and slow muscle fibers has not clearly been established. Here, we present extensive data supporting the existence of distinct functional phenotypes in mitochondria from slow and fast fibers, including differences in energy substrate preferences, regulation of oxidative phosphorylation, dynamics of reactive oxygen species release, handling of Ca2+ and regulation of cell death via the mitochondrial permeability transition pore (mPTP). The potential physiological implications on muscle function, and the putative mechanisms responsible for establishing and maintaining distinct mitochondrial phenotype across fiber types are also discussed.
Evolution of hypoxia‐responsive cytochrome c oxidase genes
Authors: Moyes Christopher , Kocha Katrinka , Pearlstein Jessica
Published: June 2021

Among the mechanisms that fine‐tune cytochrome c oxidase (COX) levels/activities is an oxygen‐dependent switch of paralogs for subunit 4. COX4‐1 is constitutively expressed in most tissues; COX4‐2 predominates in brain and lung and is induced by hypoxia in other tissues. Though there is considerable evidence that this isoform switch has advantages in COX regulation under energetic stress, the evolutionary origins of the adaptation are unclear. All vertebrates possess single gene copies of both orthologs, though birds lack a functional COX4‐2 gene. Mammals, amphibians and reptiles display considerable diversity in the distribution, degeneracy and efficacy of canonical hypoxia‐responsive elements (HRE) and oxygen‐responsive elements (ORE). Fish display mammalian‐like tissue expression profiles of COX4‐1 and COX4‐2, but we find no evidence that COX4‐2 is hypoxia responsive; fish and fish cells do not alter COX4‐2 expression under hypoxia or anoxia, and their proximal promoters lack HRE or ORE elements homologous to the human gene. These studies suggest that the COX4‐2 paralog is near ubiquitous in vertebrates, but its oxygen responsiveness is perhaps limited to mammals. Even in mammals, its oxygen sensitivity differs among tissues and between cancer lines. Funded by NSERC Canada.
Effects of angiotensin II on mitochondrial physiology in normal (PWR‐1E) and malignant (LNCaP) prostate epithelium cell lines
Authors: Duerr Jeffrey M , Kordosky Jennifer R
Published: June 2021

Prostate cancer is diagnosed in over 186,000 men in the United States and accounts for more than 28,000 deaths annually. The epidemiology of prostate cancer is identified by risk factors including genetics, diet, smoking alcohol consumption, chronic inflammation, hypertension and age. Angiotensen II (AngII) is a major effector peptide of the rennin‐angiotensin system (RAS), and is an important factor in hypertension. RAS interacts with the autocrine/paracrine system, and relates to cell growth and differentiation in some organs. Investigators have reported that exogenous application of Ang II to LNCaP cells results in the activation of Akt signaling, increased activity of SOD, and glutathione peroxidase. Another common characteristic of tumor cells is the shift away from oxidative phosphorylation to a conversion of pyruvate to lactate in the presence of ample oxygen, it remains unclear whether “aerobic glycolysis” is cause, effect, or either depending on the cancer under study. We investigated the effects of Ang II in a non‐malignant and malignant prostate cell line in order to document effects of Ang II specific to mitochondrial bioenergetics in a cancer phenotype. Our results suggest that aerobic glycolysis is induced in both cell lines, but is mediated by disparate signaling pathways.
Ethanol preconditioning augments increase in cardiac mitochondrial mass elicited by ischemia/reperfusion
Authors: Kalogeris Theodore J. , Wang Meifang , Jones Allan , Baines Christopher , Korthuis Ronald J.
Published: June 2021

Preconditioning (PC) strategies in experimental models of ischemia/reperfusion (IR) increase mitochondrial biogenesis, suggesting that increased mitochondrial mass may mediate the preconditioned phenotype. However, the effect of IR itself on mitochondrial biogenesis is not clear. In this study, we measured mitochondrial (mt)DNA as an index of mitochondrial mass, in cardiac tissue of mice subjected to either sham or 30 min occlusion of the left anterior descending coronary artery, followed by 4 h reperfusion, 24 h after gavaging with either water or ethanol (EPC, 0.61 ml/kg). Total DNA was extracted from both ventricular apex and right ventricle, and subjected to qPCR using primers for both 18s nuclear (n) and 12s mtDNA. There was little effect of IR or EPC+IR on nDNA in both cardiac apex and right ventricle. IR elicited significant increases in mitochondrial DNA (normalized to nDNA) in both cardiac apex (247 ± 21% of sham) and right ventricle (279 ± 82% of sham). MtDNA was increased even further in EPC+IR mice (apex: 407 ± 76% of sham; right ventricle: 394 ± 57% of sham). These results indicate 1) a novel, endogenous, cardiac mitochondrial biogenic response to IR, and 2) potentiation of this biogenic response by ethanol preconditioning. Thus, the beneficial effects of EPC's may be mediated, in part, through augmenting an endogenous protective mechanism related to mitochondrial biogenesis. Supported by NIH AA‐014945.
The cocoa flavanol (−)‐epicatechin exerts its cardioprotective effects by protecting myocardial bioenergetics
Authors: Yamazaki Katrina Go , Andreyev Aleksander , Petrosyan Susanna , Ceballos Guillermo , Villarreal Francisco , Murphy Anne
Published: June 2021

Targeting the mitochondria during ischemia reperfusion (IR) may confer cardioprotection and lead to improved clinical outcomes. This study tested the potential of the cocoa flavanol (−)‐epicatechin (EPI) to exert cardioprotection during IR via modulation of mitochondrial function. Ischemia was induced in rats via a 45 min occlusion. Reperfusion was allowed for 1h, 48h, or 3 wks. EPI (10 mg/kg) was administered IV 15 min prior to reperfusion for the single dose group and again 12h later for the double dose group. Controls received water. A single dose of EPI significantly reduced infarct size by 27% and 28% at 48h and 3wk, respectively, compared to controls. Double EPI treatment further decreased infarct size at 48h (80% reduction), which was sustained at 3 wk (52% reduction). In order to assess if this cardioprotection was mediated by protection of mitochondrial function, mitochondria were isolated from the left ventricle of sham, IR, and IR+EPI animals 1h after ischemia. IR animals had a significant decrease in mitochondrial O2 consumption, significant increase in mitochondrial Ca2+ levels, and decreased ATP and NADH pools. Epicatechin protected against these changes and had levels similar to sham animals. Taken together, the results suggest that EPI appears to enhance myocardial bioenergetics to confer cardioprotection. This study addresses an important topic related to the identification of mechanisms of action of an inexpensive, yet apparently effective dietary supplement for the potential treatment of cardiovascular disease. This work was supported by NIH 1R24DK092154 and Cardero Therapeutics.
Neuropeptide Y is associated with changes in appetiteassociated nuclei but not food intake in a hypophagic avian model
Authors: Newmyer Brandon Anthony , Siegel Paul B. , Cline Mark Andrew
Published: June 2021

The central mechanisms through which neuropeptide Y (NPY) exerts its potent orexigenic effect as well as potential roles for NPY in central pathways governing eating disorders have been heavily investigated since its original isolation. However, to our knowledge NPY has not been studied concurrently in polygenic models of hypo‐ and hyperphagia. Thus, this study was designed to evaluate the hypothesis that NPY is associated with differential food intake responses in lines of chickens that have undergone long‐term genetic selection for low (LWS) or high (HWS) juvenile body weight and exhibit hypo‐ and hyperphagia, respectively. LWS chicks did not respond to any dose of NPY tested (59 pmol to 5.2 nmol), while HWS chicks responded to all doses of NPY at similar magnitudes throughout the duration of observation. To examine potential differences in brain chemistries between HWS and LWS chicks after NPY administration, neuronal activity in the hypothalamus was quantified using c‐Fos immunohistochemistry. Both lines responded with similar increases in c‐Fos immunoreactivity in the lateral hypothalamus and both divisions of the paraventricular nucleus; there were no significant line or line by treatment interactions. These results support that altered body weights may be related to differential NPY‐associated food intake pathways in these lines and may have implications in the development of pharmacological options to reverse eating disorders.
Role of brain melanocortin receptor 5 in physical activity in rats
Authors: SHUKLA CHARU , NOVAK COLLEEN M
Published: June 2021

The brain melanocortin (MC) system is one of several systems that regulate energy balance; it consists of peptides such as α‐MSH that act via MC receptors (MCR). Brain MC3R and MC4R are known to modulate energy expenditure and food intake; little is known about the role of MC5R in the brain, however. We previously demonstrated that, compared to low‐activity rats, high‐activity rats show enhanced MC5R mRNA expression in the peri‐fornical lateral hypothalamus (PeFLH) but not in the paraventricular nucleus (PVN). To test the hypothesis that hypothalamic MC5R expression is related to enhanced physical activity (PA), we stereotaxically implanted guide cannulae in male Sprague‐Dawley rats, then measured the ability of a novel MC5R‐specific agonist in the PVN and PeFLH to increase PA. Intra‐PeFLH microinjections significantly enhanced PA in a dose‐dependent manner, while no such effect was seen in the PVN. The MC5R agonist (compared to vehicle) did not significantly suppress food intake in either of the hypothalamic regions, however. Thus, specific, regional activation of brain MC receptors could affect different aspects of energy balance, particularly PA. We will now investigate if these site‐ and receptor subtype‐specific MC differences may underlie individual differences in daily PA. MC5R agonist was obtained from M. Bednarek (MedImmune Ltd, Cambridge, UK). Research support was from NIH R01NS055859 to CMN and AHA11PRE7320029 to CS.
Multiple subtypes of opioid receptors regulate binge‐like (BE) feeding in rodents on an intermittent access schedule
Authors: Statnick Michael A , Sahr Allison E , Pintar John , Czyzyk Traci
Published: June 2021

Binge eating disorder (BED) affects approximately 3% of adults in the USA and is characterized by repeated episodes of eating unusually large amounts of food in a short period of time without engaging in compensatory behaviors. Interestingly, a significant comorbidity with substance abuse exists in patients with BED. Opioid peptides are known to regulate appetitive behavior. Moreover, opioid receptor antagonists (OpRA) are used clinically in the treatment of alcohol and substance abuse. In both rats and mice, the pan OpRA LY255582 was effective in reducing BE at doses that did not affect control 24 hour intake. In rats, similar reductions in BE were observed using the mu and delta selective OpRAs LY2491481 and naltriben, respectively. The kappa selective OpRA LY2444296 was ineffective in reducing BE in rats. In order to confirm our pharmacology findings using genetic models, we evaluated BE in OpR knockout mice using a similar intermittent access paradigm. Unlike our antagonist studies, no differences in BE were observed in either the mu or delta receptor mutants compared to wildtypes. Interestingly, spontaneous free access feeding and BE were increased in the kappa receptor specific and mu‐delta‐kappa combinatorial mutants in comparison to wildtypes. Our data support that endogenous opioids modulate BE in a complex manner in rodents.
Orexigenic and obesity effects of estrogen deficiency can be attenuated by half‐day food deprivation either during the light phase or the dark phase
Authors: Taguchi Sayumi , Mabuchi Kaori , Takano Azusa , Hara Yayoi , Morimoto Keiko , Takamata Akira
Published: June 2021

Estrogen deficiency enhances food intake, especially during the light phase, and also body weight gain. In the present study, we examined the effect of food deprivation during the light phase and the dark phase on the estrogen deficiency‐induced enhancement of daily food intake and body weight gain. Ovariectomized rats were assigned into the estrogen (E2)‐treated or vehicle (Veh)‐treated group. Rats were then fed ad libitum or with food deprivation either during the light phase or the dark phase. At the end of the experiment, we examined the number of c‐Fos, Bmal1 and Per1 reactive cells in the SCN during the light and dark phases. Daily food intake, total food intake over 2 weeks and body weight gain were not affected by the diurnal food deprivation in the E2 group, but these were attenuated in the Veh group. Food deprivation during the dark phase reduced daily food intake and body weight gain in the both of E2 and Veh groups compared with ad lib feeding, the decrease was larger in the Veh group than the E2 group, and daily food intake became similar between the groups. Expressions of c‐Fos and Bmal1 in the SCN were not significantly different between the two groups either during the light or dark phase. These data indicate that orexigenic effect of estrogen deficiency can be abolished by half‐day food deprivation either during the light phase or the dark phase, and the SCN is not likely involved in the regulations of daily food intake and body weight in response to the half‐day food deprivation.
Transcription Factors and DNA Regulatory Elements
Authors: Bulyk Martha L.
Published: June 2021

The interactions between sequence‐specific transcription factors (TFs) and their DNA binding sites are an integral part of the gene regulatory networks within cells. My group developed highly parallel in vitro microarray technology, termed protein binding microarrays (PBMs), for the characterization of the sequence specificities of DNA‐protein interactions at high resolution. Using PBMs, we have determined the DNA binding specificities of hundreds of TFs from a wide range of species. More recently we have used the PBM technology to investigate TF heterodimers and higher order complexes. The PBM data have permitted us to identify novel TFs and their DNA binding sequence preferences, predict the target genes and condition‐specific regulatory roles of TFs, predict and analyze tissue‐specific transcriptional enhancers, investigate functional divergence of paralogous TFs within a TF family, investigate the molecular determinants of TF‐DNA recognition specificity, and distinguish direct versus indirect TF‐DNA interactions in vivo. Notably, not all DNA binding sites of a TF function equally. Further analyses of TFs and cis regulatory elements are likely to reveal features of cis regulatory sequences that are important in gene regulation.
Angiotensinergic signaling in the brain controls whole‐body metabolism: Role of adipose AT2 receptors
Authors: Grobe Justin L , Park Sungmi , Liu Xuebo , Sigmund Curt D
Published: June 2021

Transgenic “sRA” mice exhibit elevated brain renin‐angiotensin system (RAS) activity through expression of a human angiotensinogen transgene and a neuron‐specific human renin transgene. sRA mice exhibit elevated metabolic rate, partially mediated through a suppression of the circulating RAS. Uncoupling protein‐1 (UCP1; 24.6‐fold, n=5, P<0.01), and PPARγ‐coactivator‐1α (PGC‐1α; 2.8‐fold, n=5, P=0.01) mRNA were increased selectively in subcutaneous adipose (scWAT) of sRA mice, suggesting an increased brown adipocyte content. Treating differentiated 3T3‐L1 preadipocytes with the AT2 antagonist PD‐123,319 induced PGC1α (2.4‐fold) and the PGC1α target, catalase (3.4‐fold), while the AT1 antagonist Losartan had no effect. Treating sRA mice with the AT2 agonist CGP‐42112a (90 ng/hr, s.c., 8 weeks) normalized scWAT UCP1 mRNA (vehicle 24.6 vs CGP 2.1‐fold of control, n=5,7, P<0.05), suppressed resting metabolic rate (3.59±0.22 vs 3.12±0.17 mL O2/100g/min, P<0.01), and increased body weight (17.8±0.5 vs 23.1±2.1 grams, P=0.01), but had no effect on food intake (3.1±0.2 vs 3.6±0.3 g/day, P=0.72). These data support a model where AT2 receptor‐mediated suppression of adipose transdifferentiation mediates increased thermogenic capacity in animals with elevated brain RAS activity. Selective antagonism of AT2 receptors in adipose tissue may therefore represent a novel therapeutic strategy for obesity.