bluuebunny/crossref_metadata_2025 huggingface dataset:
Steric Gate Variants in a Y family DNA Polymerase Confer UV‐Hypersensitivity
Authors: Beuning Penny J , Shurtleff Brenna W , Sefcikova Jana , Walker Graham C
Published: June 2021

Induction of the SOS response due to a cell's failed attempts to replicate damaged DNA results in the expression of at least 43 genes in E. coli. The induced genes include umuDC, whose products include the Y‐family DNA polymerase DNA pol V (UmuD′2C). E. coli UmuC is a DNA polymerase with the specialized ability to copy past lesions in the DNA template, but this activity may be mutagenic. Normal, replicative DNA polymerases have a “steric gate” residue that prevents incorporation of ribonucleotides in the nascent DNA, by serving as a steric block to the 2′‐OH. In the Y‐family polymerases, this residue appears important for facilitating lesion bypass activity. We have found that steric gate mutants of UmuC cause extreme cellular sensitivity to UV exposure and are dominant negative at low copy number. Mutations in the known beta clamp binding motifs of UmuC or deletion of the proofreading subunit of DNA pol III suppress the UV hypersensitivity of the steric gate variant. We have found that mutation of residues surrounding the steric gate result in decreased mutagenesis without cellular hypersensitivity to UV light.

Supported by a Dreyfus Foundation New Faculty Award.
Understanding the Role of Phosphorylation of the Upf Proteins in the Activity of the Nonsense‐Mediated mRNA Decay (NMD) Pathway
Authors: Lasalde Clarivel , Cajigas Iván J , León Alfredo , González Rosalyn , Cardé‐Serrano José , Vega Irving E , González Carlos I
Published: June 2021

Nonsense‐mediated mRNA decay (NMD) is an mRNA surveillance mechanism that degrades transcripts harboring premature termination codons (PTCs), preventing the synthesis of potentially deleterious proteins. Upf1p, Upf2p, and Upf3p are central components of the NMD pathway in Saccharomyces cerevisiae. Upf1p and Upf2p have been shown to be phosphorylated both in yeast and humans. Specifically, phosphorylation of the N‐terminal region of Upf2p is crucial for its ability to elicit NMD and for its interaction with Hrp1p, another component of this mRNA surveillance pathway. Previous studies have demonstrated that Upf1p phosphorylation is necessary for activation of the NMD pathway in C. elegans and humans. We have recently identified eleven phosphorylation sites in yeast Upf1p using mass spectrometry. Some of the phosphorylated residues in yeast Upf1p are evolutionary conserved in humans, Drosophila, Arabidopsis and C. elegans. Notably, mutations in some of these conserved phosphorylated residues disrupt the activity of the NMD pathway. We are currently conducting similar mass spectrometry analysis in yeast Upf3p. All together, these studies will allow us to better understand the role of phosphorylation in the activity of the NMD mRNA surveillance pathway in eukaryotes. Supported by RISE 2R25GM61151, NIH U54 CA96297–03, NIH‐NIGMS SCoRE GM008102–305, NIH‐NCRR P20RR016174, HRD‐01843,FIPI‐UPR.
Differences in electrogenic secretion of electrolytes in colonic epithelium of inbred mouse strains.
Authors: Flores Carlos A , Sepulveda Francisco V , Cid L Pablo
Published: June 2021

The mouse is the animal model of choice in studies of the physiology and pathophysiology of intestinal fluid and electrolyte transport. In the course of our research in colon physiology we have observed differences in electrical tissue properties and responses to agonists that induce electrogenic secretion of electrolytes among mice strains. Such differences are important enough to preclude comparisons between strains and might be crucial when choosing the genetic background for knockout of genes of interest. We have now investigated some of these differences by electrophysiological methods. Colonic tissue from Black Swiss, 129Sv and B6 mice were placed in Ussing chambers and their responses to a series of agonists and blockers recorded. We observe that while the 3 strains have similar anion secretory responses to increasing cAMP (around ‐100 μA cm−2), when the muscarinic agonist carbachol was used the largest response was seen with B6 tissue, compared to that of Black Swiss and 129Sv colon (‐200, ‐163 and ‐133 μA cm−2 respectively). The importance of the genetic background on the severity of cystic fibrosis in mice has long been known (Rozmahel et al., Nat Genet 12: 280‐7, 1996) and has been related to differences in calcium‐dependent anion secretion. We reported previously that basolateral KCNN4 calcium‐activated potassium channels are the rate‐limiting step in calcium‐agonist‐dependent anion secretion, rather than apical CFTR chloride channels (Flores et al., J Physiol 583: 705‐17, 2007). We are studying KCNN4 expression as a possible explanation to these differences.

Supported by FONDECYT 3085023
Genome‐wide RNA interference screen for phosphatases that contribute to regulation of the transcription factor TonEBP/OREBP
Authors: Zhou Xiaoming , Ferraris Joan D. , Burg Maurice B.
Published: June 2021

High NaCl activates TonEBP/OREBP, resulting in increased transcription of osmoprotective genes. Regulation of protein phosphorylation is known to be involved. Some of the kinases are known, but the phosphatases are not. In the present studies we screened a genome‐wide human phosphatase siRNA library in HEK293 cells for effects on TonEBP/OREBP transcriptional activity. siRNAs against 57 phosphatases significantly alter TonEBP/OREBP transcriptional activity at 500 mosmol/kg or 290 mosmol/kg or both. At 500 mosmol/kg, 31 siRNAs increase it and 16 decrease it. At 290 mosmol/kg 15 siRNAs increase it and 4 sets decrease it. 4 of the siRNAs increase TonEBP/OREBP activity at both osmolalities, 3 decrease it and 2 have opposite effects. Also, 9 siRNAs augment it at 290 mosmol/kg, but not at 500 mosmol/kg. The phosphatases that are identified include ones previously known to affect cell cycle, cytoskeleton, or MAPKs, and metabolism of phospholipids, RNA or glucose. Among 31 siRNAs that increase TonEBP/OREBP activity at 500 mosmol/kg, we have confirmed the effect of PPP1R3C and SHP‐1 by overexpression of these proteins. Additional studies are required to decide whether the known actions of the phosphatases explain the effects on TonEBP/OREBP or whether novel mechanisms are involved. We conclude that TonEBP/OREBP is regulated by phosphatases, most of which reduce its activity. Supported by the Intramural Program of NHLBI.
Connexin 40 is essential for hypoxic pulmonary vasoconstriction
Authors: Wang Liming , Isakson Brant , de Wit Cor , Kuppe Hermann , Kuebler Wolfgang M
Published: June 2021

Hypoxic pulmonary vasoconstriction (HPV) has traditionally been attributed exclusively to O2 sensing in pulmonary vascular smooth muscle cells (PASMC). Yet, the predominant site of gas exchange, and hence, the ideal site for O2 sensing is in pulmonary capillaries, where PASMCs are absent. The spatial separation between a capillary O2 sensor and PASMC in upstream arterioles would require a retrograde propagation of signals along the endothelial layer. Connexin 40 (Cx40) is expressed in lung endothelial cells and mediates conducted responses in the systemic circulation. Here, in isolated mouse lungs, we tested our hypothesis that Cx40 plays a critical role in HPV. HPV was quantified as increase in pulmonary artery pressure (PAP) in response to hypoxic ventilation (1% FiO2). Intrinsic pulmonary vascular resistance (R0) was determined by nonlinear regression analysis of pressure‐flow curves. Hypoxia‐induced increases in PAP and R0 were attenuated by 75% in lungs perfused with the nonspecific gap junction blocker 18α‐glycyrrhetinic acid, by 55% with Cx40 specific mimetic peptide Gap2740, and by 64% in Cx40−/− mice. The role of Cx40 is specific for HPV, since the vasoconstriction response to angiotensin II remained unaffected in all groups. Our data demonstrate a critical role of Cx40 in HPV, and suggest that microvascular endothelial cells may propagate the hypoxic signal to upstream arterioles via endothelial Cx40.
Chronic cannabinoid administration increases CD8+ lymphocyte apoptosis during early Simian Immunodeficiency Virus infection in rhesus macaques.
Authors: Walker Edith , Birke Leslie , Vande Stouwe Curtis , Zhang Ping , Amedee Angela , Winsauer Peter , Molina Patricia
Published: June 2021

Δ9‐tetrahydrocannabinol (Δ9‐THC), the primary psychoactive component of marijuana, is used to ameliorate Acquired Immune Deficiency Syndrome associated wasting. Cannabinoid receptors are expressed in cells of the immune system, raising the possibility that chronic Δ9‐THC use may impact on immune function, potentially affecting progression of human immunodeficiency virus infection. We examined the effects of chronic Δ9‐THC administration (0.32mg/kg i.m., 2 X daily, starting 30 d pre‐infection) on the early changes in lymphocyte counts, subpopulations, apoptosis, and proliferation in young adult male rhesus macaques at day 30 post simian immunodeficiency virus (SIVmac251; 100 TCID50/ml, i.v.) infection. SIV+ animals had lower (40%) lymphocyte CD4+/CD8+ ratio, 3‐7‐fold higher CD8+Ki67 expression (proliferation marker), without significant changes in total lymphocyte count, or CD4+ and CD8+caspase 3 expression (apoptosis marker) than time‐matched SIV‐ controls. THC‐treated SIV+ animals showed similarly decreased CD4+/CD8+, increased CD8+Ki67 expression, but higher expression of caspase 3 in CD8+ lymphocytes than time‐matched THC‐treated SIV‐ controls. We speculate that though modest the Δ9‐THC‐induced increase in CD8+ apoptosis may accentuate the CD4+/CD8+ imbalance during later stages of SIV infection. Supported NIDA‐020419‐01A1
LPS and hypoxia alters the production/release of catecholamines in Immortalized Human Mesangial Cell
Authors: Zanini Ana Paula , Arita Danielle Yuri , Almeida Waldemar Silva , Casarini Dulce Elena
Published: June 2021

Catecholamines (CA)‐dopamine (DA), L‐Dopa, epinephrine (EP) and norepinephrine‐are involved in filtration and renal vasoconstriction processes, and can be produced by mesangial cells (MC). MC expresses Toll Like Receptor 4, that detects lipopolysaccharide (LPS). LPS and hypoxia, both present in sepsis, can lead to acute renal failure. The aim was to asses the effect of LPS and hypoxia on the production/release of CA in immortalized human mesangial cell (IHMC). IHMC were cultured in DMEM and incubated with LPS (0.1, 1.0, 5.0 and 100 μg/mL) for 2 hours, or exposed to hypoxia for 30 min. CA were extracted from the medium and cell lysates was quantified by HPLC‐ED. Tyrosine hydroxylase (TH) expression ‐ step‐limiting enzyme ‐ was evaluated by Western blotting. LPS at 1 μg/mL induced a decline in intracellular L‐Dopa level as compared with control (5.3±1.6 vs.10.8±1.0 pg/mg protein). The concentration of 5 μg/mL decreased the intracellular level of L‐Dopa (4.9±1.3 vs. 10.3±1.0 pg/mg protein) and increased EP concentration (8.8±1.9 vs. 2.2±0.5 pg/mg protein). LPS at 100 μg/mL increased the levels of intracellular EP (102.7±8.4 vs.65.8±10.1 pg/mg), L‐Dopa (48.5±2.4 vs.22.2±4.1 pg/mg protein) and DA (24.9±2.9 vs.15.9±1.4 pg/mg protein) and decreased the concentration of EP (125.2±13.7 vs.205.4±10.4 pg/mg protein) and L‐Dopa (34.6±5.7 vs.57.4±8.6 pg/mg protein) in extracellular. TH expression was significantly affected by LPS (100 μg/ml), however it did not occurr under hypoxia condition. These results suggest that LPS can influence the production/release of CA in IHMC.

Support by CNPq.
The Influence of Tungstate‐Exposure on Immunological Response to RSV Infection in C57BL/6 Mice
Authors: Fastje Cynthia Darlene , Harper Kevin , Park Yong Soo , Wong Simon S. , Witten Mark L.
Published: June 2021

Significantly elevated concentrations of sodium tungstate have been demonstrated in the atmospheric particulate matter in multiple communities experiencing elevated rates of childhood leukemia, but not in their respective control communities. Prenatal exposure to sodium tungstate in C57BL/6 mice significantly decreased the transcriptome expression of Dmbt1, a protein which functions to aggregate bacteria and viruses in lung mucosa and saliva. Additionally, network analysis of gene microarray data produced a significant network associated with hematological/immunological disease focused on genes involved in viral defense. We hypothesize that exposure to sodium tungstate alters immunological and hematological response to viral infection as measured by qRT‐PCR of RSV genes F & G during the fastigium of the infection, CBC with a differential, and RT2‐PCR for genes associated with immunological function. C57BL/6 mice, exposed prenatally to sodium tungstate, demonstated a differential response to RSV viral infection. Additionally, tungsten exposure in male mice has a deleterious effect on testes function.

Supported by EPA Grant EM‐96963201
Autonomic Dysregulation in a Mouse Model of Muscular Dystrophy Destined to Develop Dilated Cardiomyopathy
Authors: Sabharwal Rasna , Weiss Robert M , Chapleau Mark W
Published: June 2021

Mutations in the dystrophin‐glycoprotein complex cause muscular dystrophy and dilated cardiomyopathy (DCM) in animals and humans. Mice deficient in sarcoglycan delta (Sgcd), a component of this complex, develop muscular dystrophy at a young age (<10 wks) with later onset of DCM (Cell 98:465‐474,1999). We hypothesized that autonomic dysregulation may occur early in the disease progression before development of DCM. We measured cardiac function (echocardiography); blood pressure (BP) and heart rate (HR) (telemetry); and indices of autonomic regulation including baroreflex sensitivity (BRS) and HR variability (HRV‐SD) in 10‐12 wk old Sgcd‐/‐ and control C57BL/6 mice. Left ventricular ejection fraction (0.83±0.01 vs. 0.85±0.03) and end‐diastolic volume (26±2 vs. 33±4 µl) were normal in Sgcd‐/‐ mice. Histopathological features of muscular dystrophy were evident in skeletal muscle but not in the heart of Sgcd‐/‐ mice. Compared with control mice, Sgcd‐/‐ mice exhibited less locomotor activity (4±1 vs. 11±1 c/min), lower mean BP (100±2 vs.120±4 mmHg), and reductions in diurnal (night‐day) variability, BRS and HRV‐SD (see Table, *P<0.05).

We conclude that young Sgcd‐/‐ mice exhibit decreased BP, loss of diurnal cardiovascular rhythms, and severe autonomic dysregulation prior to onset of cardiac dysfunction. We speculate that this early autonomic dysregulation may hasten the progression to DCM. (VA)
Sympatho‐inhibitory effect of atorvastatin due to anti‐senescence and attenuation of impaired neurogenesis in RVLM of SHRSP
Authors: Kishi Takuya , Hirooka Yoshitaka , Ito Koji , Araki Shuichiro , Konno Satomi , Sunagawa Kenji
Published: June 2021

Background

Neural senescence in brain is progressed by reactive oxygen species (ROS), Ras‐caspase 3‐induced apoptosis and abnormal shortening telomere length, and is associated with neural function. Atorvastatin inhibits sympathetic nerve activity (SNA) by reduction of ROS in rostral ventrolateral medulla (RVLM) of stroke‐prone spontaneously hypertensive rats (SHRSP). Neurogenesis is reported to be impaired in RVLM of SHRSP. We hypothesized that atorvastatin inhibits SNA due to anti‐senescence and attenuation of impaired neurogenesis in RVLM of SHRSP.

Methods and Results

In age‐matched control SHRSP (CNT), SNA, ROS, Ras activity, caspase‐3, p53 and p21 and neural senescence determined by β ‐galactosidase staining were significantly higher and telomere length was significantly shorter than in WKY (n=5 for each). In atorvastatin‐treated SHRSP (20 μg/kg/day, intracerebroventricular injection for 14 days, n=5) (ATORVA), SNA, ROS, Ras activity, caspase‐3, p53 and p21 and neural senescence were all inhibited and telomere length was significantly longer than in CNT. Neurogenesis in RVLM was significantly impaired in CNT than in WKY, and significantly improved in ATORVA than in CNT.

Conclusion

Atorvastatin attenuates neural cell senescence and impaired neurogenesis in RVLM of SHRSP, which contribute to sympatho‐inhibitory effect of atorvastatin.
Prenatal nicotine exposure exaggerates thermal prolongation of the laryngeal chemoreflex in rat pups
Authors: Xia Luxi , Leiter James C. , Bartlett Donald
Published: June 2021

Laryngeal chemoreflex (LCR) apnea occurs in infants in response to water in the larynx and may contribute to pathogenesis of SIDS. The LCR is prolonged in neonatal piglets and rats that are warmed 1‐3 degrees C, and this thermal prolongation is exaggerated in rat pups born to mothers exposed to cigarette smoke during pregnancy. Since smoking mothers expose their fetuses to nicotine, we studied the effect on the LCR of combined prenatal nicotine exposure and brief postnatal hyperthermia in anesthetized rat pups. Pregnant rats were exposed to nicotine (6.4 mg/kg/day) or saline by infusion via implanted osmotic minipumps from gestational day 3 (GD3) until GD 21. The LCR was assessed between postnatal days 4‐15 with and without hyperthermia. Prenatal nicotine exposure exaggerated the thermal prolongation of the LCR. In the nicotine group (n=21), respiratory disruption by the LCR during hyperthermia averaged 13.24 1.85 sec, and the longest apnea averaged 8.29 1.29 sec. In the control group (n=24), respiratory disruption under the same conditions averaged 7.19 0.97 sec, and the longest apnea averaged 4.68 0.76 sec. Thus, prenatal nicotine exposure enhances the hyperthermic prolongation of the LCR, which has previously been shown in piglets to depend on GABAergic inhibition of breathing.

(Supported by P01‐HD36379, HD042707 and a grant from the Flight Attendants Medical Research Institute.)
Recruitment Order of Diaphragm Motor Units During Different Respiratory Behaviors
Authors: Seven Yasin Baris , Zhan Wen‐Zhi , Sieck Dylan C. , Sieck Gary C. , Mantilla Carlos B.
Published: June 2021

In skeletal muscles, force gradation depends on the level of motor unit (MU) activation. MU recruitment generally follows a specific order, with MUs comprising type I or type IIa fibers more active than type IIx and/or IIb. Direct evidence of MU recruitment across motor behaviors is limited to samples of relatively few MUs within a pool. The purpose of this study was to determine the extent of diaphragm MU recruitment across different motor behaviors. We hypothesized that in vivo labeling of individual neuromuscular junctions (NMJs) by FM4‐64 reflects the activation of MUs. Adult rats were exposed to eupneic (E), hypoxic/hypercarbic (H) conditions or supramaximal phrenic nerve stimulation (S). Individual diaphragm NMJs were type‐identified by myosin heavy chain expression. Average FM4‐64 labeling for NMJs at type I or IIa fibers vs. type IIx and/or IIb fibers increased progressively from E to H to S. The proportion of labeled NMJs of each type also increased significantly. At type I or IIa fibers, 40%, 59% and 90% of NMJs were labeled during E, H and S, respectively. At type IIx and/or IIb fibers, 21%, 32% and 82% of NMJs were labeled during E, H and S, respectively. Thus, using an in vivo labeling method there is direct evidence of progressive MU recruitment across different ventilatory and non‐ventilatory behaviors in the rat diaphragm.

Supported by NIH grants HL37680 and AR51173, and the Mayo Clinic.
Dual effects of ATP and neuronal NOS inhibition in the NTS neurons
Authors: Granjeiro Erica M , Accorsi‐Mendonça Daniela , Bonagamba Leni GH , Leão Ricardo M , Machado Benedito H
Published: June 2021

The cNTS is the site of synaptic relay of peripheral chemoreceptor afferents and ventral medulla‐projecting NTS neurons are related to respiratory and autonomic responses to chemoreflex. It has been suggested that ATP and L‐glutamate are co‐transmitters in the NTS and NO can modulate glutamatergic and purinergic effects in the brain. Here, we evaluated the effect of nNOS inhibition on the responses to ATP application in the NTS using awake rats and brainstem slices. In a group of awake rats the microinjection of N‐propyl‐L‐arginine, a nNOS inhibitor, into cNTS significantly reduced the increase in respiratory frequency (65 ± 10 vs 15 ± 5 cpm; n=7) produced by microinjection of ATP (1.25 mM). Another group of rats received DiI tracer into ventral surface of medulla and 24 hours later the labeled ventral surface‐projecting NTS neurons were identify in transversal slices. Glutamatergic spontaneous and TS‐evoked excitatory pos‐synaptic currents (sEPSCs and TS‐eEPSCs respectively) were recorded using whole cell patch clamp. ATP (500 μM) increased the frequency of sEPSCs (0.96 ± 0.4 vs 4.9 ± 1.3 Hz, n=8), but decreased the peak amplitude of TS‐eEPSCs (115 ± 24 vs 68 ± 13 pA, n=10). N‐propyl‐L‐arginine (60 μM) reduced only the ATP effect in the sEPSCs frequency (4.9 ± 1.3 vs 1.99 ± 0.96 Hz). The data show that: (1) the respiratory response to ATP microinjection in awake rats is affected by inhibition of nNOS; (2) ATP has a dual effect on NTS neurons; (3) inhibition of nNOS affected only the sEPSCs frequency. We concluded that the effects of ATP in the NTS neurons may involve NO production. Supported by CNPq and FAPESP.
Tissue distribution of a novel non‐AT1, non‐AT2 binding site for angiotensins in the mouse
Authors: Rabey Felicia M , Karamyan Vardan T , Speth Robert C.
Published: June 2021

We recently reported the existence of a novel, non‐AT1, non‐AT2 binding site for angiotensins II (Ang II) and III (Ang III) in the rat, mouse and human brain. While the absence of this binding site in the liver, adrenal, and kidney suggested that this binding site was brain‐specific, we now report its presence in other mouse tissues. 125I‐Sar1,Ile8 Ang II binding was determined as described previously (Karamyan et al., Eur J. Pharmacol., 2008, 590: 87) in tissue homogenates with or without 0.3 mM parachloromercuribenzoate (PCMB), in the presence of 10 μM losartan and 10 μM PD123319 to saturate AT1 and AT2 receptors, respectively. In the absence of 0.3 mM PCMB specific (3 μM Ang II displaceable) binding of 125I‐ Sar1,Ile8 Ang II could not be measured reliably. In the presence of 0.3 mM PCMB, saturable, high affinity binding was observed in the testis (4.1±2.2 pmol/g, KD = 5.0±2.7 nM) with comparable density to the brain (4.2±2.0 pmol/g), but slightly lower affinity (KD = 2.7±1.2 nM, p<0.05). Measurable binding was present in 5 of 6 pancreas and 4 of 7 spleen samples with KD values similar to the brain, but at lower (23 and 30%, respectively) binding density. Binding was generally undetectable in the lung and heart. The presence of this binding site in testis, pancreas and spleen may indicate additional functions for angiotensins in these tissues. Supported by the Peptide Radioiodination Service Center of the University of Mississippi.
Exogenous bilirubin administration exerts renoprotective effects in angiotensin II‐hypertension
Authors: Botros Fady T. , LeBlanc Ryan M. , Navar L. Gabriel
Published: June 2021

Bilirubin is an endogenous antioxidant and is the end product of heme catabolism by heme oxygenase (HO) and biliverdin reductase. Chronic angiotensin II (Ang II) infusion induces renal HO‐1 expression which is associated with renoprotective effects, and further induction of renal HO‐1 attenuates the development of hypertension in this model. To determine the effects of bilirubin on the development of Ang II‐induced hypertension, two groups of rats were studied: Ang II (n=4) and Ang II+Bilirubin (n=4). Rats were infused with Ang II (80 ng/min for 2 weeks) and bilirubin was administered simultaneously (3mg/100g BWt/48 hrs, intraperitoneally). Two weeks after Ang II infusion, systolic blood pressure significantly increased from 134±4 mmHg to 198±7 mmHg (p<0.05) in the Ang II group, and from 128±8 mmHg to 209±9 mmHg (p<0.05) in the Ang II+Bilirubin group. Relative to the Ang II group, treatment with bilirubin did not alter body weight 259±18 g vs. 275±18 g, food intake 25±3 g vs. 25±2 g, water intake 78.1±6.7 vs. 75.3±7.3 ml/day, or urine output 58.5±5.8 ml vs. 50.8±5.5 ml/day. However, urinary protein excretion was significantly lower in the Ang II+Bilirubin group (32.9±9.7 mg/day vs. 81.4±22.8 mg/day, p<0.05). We conclude that bilirubin contributes to the renoprotective effects conferred by induction of HO‐1 in Ang II‐dependent hypertension. Supported by NIH grants HL26371 and COBRE P20 RR 017659.
Hypertension induced by chronic intermittent hypoxia is accompanied by reduced expression of neuronal nitric oxide synthase (nNOS) and angiotensin type 1 receptors (AT1R) in the hypothalamic PVN
Authors: Carreño Flávia Regina , Calderon Alfred S , Ji Lisa , Mifflin Steven W , Toney Glenn M , Cunningham J Thomas
Published: June 2021

Chronic intermittent hypoxia (CIH) is an animal model of the hypoxemia associated with sleep apnea that is associated with hypertension related to increased sympathetic outflow and activation of the renin‐angiotensin system. This study tested effects of CIH on nNOS and AT1R abundance in the paraventricular nucleus (PVN) and nucleus of the solitary tract (NTS). Adult male rats were exposed to CIH (between 8am and 4pm alternate 21% and 10% O2 every 3 min) for 7 days. Previous studies have shown that such exposures to CIH significantly elevate blood pressure. On the 8th day, rats were anesthetized, their brains were removed, and punch samples containing the PVN and NTS were collected. Punches were used for Western Blot analysis for nNOS, AT1R and β‐actin. CIH had no significant effect on nNOS or AT1R expression in the NTS. However, both nNOS and AT1R expression were reduced in the PVN ofCIH rats compared to controls. A separate RT‐QPCR analysis demonstrated a decrease (~15%) in AT1aR mRNAin the PVN of CIH rats. The results obtained from this study suggest that the down‐regulation of nNOS expression in the PVN may contribute to the central mechanism that produce increased sympathetic outflow and hypertension during CIH. Decreased AT1 receptor expression in the PVN could be a compensatory response related to increased activation. P01 HL088052
SPOO2P, a botanical nutriceutical, inhibits in vitro aortic contractions and lowers blood pressure of anesthetized hypertensive rats
Authors: Joshua Irving Gilbert , Adeagbo Ayotunde , Patricia Madjani , Iwu Evangrline , Inderjet Dev , Subbiah Ven
Published: June 2021

Elevated endothelin levels are associated with cardiovascular diseases, e.g., hypertension and chronic heart failure. Development of an endothelin receptor inhibitor is potentially beneficial for treating some cardiovascular diseases. SPOO2P (PhytoPharmacon, LLC, Durham, NC) is a purified botanical extract, containing both myriceric acids A and C. Myriceric acid A has been reported as a non‐peptide antagonist of endothelin A (ETA) receptors. The present study determined the effects of SPOO2P on in vitro aortic muscle contractions and on blood pressure of anesthetized rats. Contraction of aortic rings bathed with physiologic salt solution (37°C; gassed with carbogen) was measured with force displacement transducers coupled to Tissue Force Analyzers (Micromed, Louisville,KY). Hypertension was induced with nitro‐L‐arginine methyl ester (L‐NAME) administered in drinking water (1g/L) for 3 weeks. Topical SPOO2P (20‐100 µg/ml) antagonized aortic contractions elicited by endothelin‐1, phenylephrine, serotonin, KCl and NaF over in a dose dependent manner with maximal inhibition ranging from 60‐90%. The compound (100 mg/kg; oral gavage) also significantly reduced the mean arterial blood pressure (MAP) of control and L‐NAME hypertensive rats. We conclude that SPOO2P is a potentially useful anti‐hypertensive nutriceutical. [Support: University of Louisville Research Initiation Grant].
Effects of Acute Stress on Blood Pressure, Catecholamines, and Renal Interlobar Artery Constriction in Spontaneously Hypertensive (SHR) and Normotensive (WKY) Male Rats
Authors: Peck Jennifer , Novak Jacqueline , Hover Jeffery , Reho John , Dunphy Gail , Boehme Shannon , Ely Daniel , Ramirez Rolando
Published: June 2021

Human cardiovascular diseases are often preceded by and exaggerated with stress, affecting blood pressure, catecholamines (CATS), and vessel reactivity. To investigate these mechanisms we used acute restraint stress and measured changes in: systolic blood pressure (SBP), plasma and urinary CATS, and responses of isolated renal interlobar arteries to the vasoconstrictor phenylephrine (PE). We hypothesized that stress would increase sympathetic nervous system (SNS) response by elevating SBP, both plasma and urinary CATS, and increase constriction of renal interlobar arteries. Adult male SHR rats were subjected to 4 weeks of 1hr restraint stress (n=8, 3dys/wk) compared to non‐stressed SHR (n=6) and WKY (n=6) controls. Data pending for stressed WKY males. Blood pressure was measured weekly via tail cuff, and CATS were assayed by HPLC. Small renal interlobar artery constriction of response to PE was determined using a pressurized arteriograph system. Stressed SHR animals exhibited increased plasma and urinary CATS. Constrictor responses to PE in stressed animals were decreased when compared to control animals, for example at 1.0mmol PE stressed SHRs demonstrated 48% maximum constriction while controls showed 80.3% (SHR) and 75.3% (WKY). In conclusion, acute stress does increase SNS activity through plasma CATS, but did not increase SBP. Vasoconstrictor responses of isolated arteries appear to be increased.
Bioimpedance measurements to determine fluid volume distribution in desoxycorticosterone acetate (DOCA)‐salt hypertension
Authors: Seitz Bridget Mahon , Fink Gregory
Published: June 2021

Splanchnic arterial and venous constriction promotes redistribution of blood volume from the abdominal region into the central circulation and thereby into less compliant vascular beds. We hypothesized that this mechanism contributes to the development of DOCA‐salt hypertension. Bioimpedance was used to estimate blood volume translocation in rats by permanently implanting electrodes in pairs across the chest and abdomen. Radiotelemetry transmitters were also surgically implanted to simultaneously measure blood pressure. In addition, all animals underwent a uninephrectomy and a control period of distilled water intake. Some rats were switched to high salt water for 3 days and then implanted with a DOCA pellet. After 5 days DOCA rats were then switched back to distilled water. Arterial pressure did not change during the control or high salt intake alone. Administration of DOCA to rats on high salt intake caused a slow increase in arterial pressure. There were no changes in impedance in the thoracic region in either group of rats during the study. However, there was a two‐fold increase in impedance (most likely indicating decreased blood volume) in the abdominal region during the onset of DOCA‐salt hypertension, which was reversed when rats were given distilled water. We conclude that blood translocation from the abdominal region may participate in the development of DOCA salt hypertension.
Bestrophin‐3‐associated Ca2+‐activated Cl− channel in vascular smooth muscle and endothelial cells
Authors: Matchkov Vladimir V. , Eriksen Tina Fransgaard , Larsen Per , Pedersen Finn Skou , Nilsson Holger , Aalkjaer Christian
Published: June 2021

We have recently shown (1) that bestrophin‐3, a member of the Best family, is essential for the cGMP‐dependent Ca2+‐activated Cl− current (I(Ca, cGMP)Cl) (2) which has been shown in different types of smooth muscles (3). The I(Ca, cGMP)Cl has similar characteristics as the currents produced by heterologous expression of bestrophins and distributs similarly to Best‐3 expression. Furthermore, downregulation of Best‐3 with siRNA was associated with a significant reduction of the I(Ca, cGMP)Cl.

To study the physiological significance of Best‐3 immunohistochemical (IHC) and siRNA approaches were used. Wistar rats were used in accordance with national guidelines for animal research.

We characterized the Best‐3 specific antibody by expression of eGFP‐fusion‐protein with the epitope containing peptide. IHC showed a broad expression of Best‐3 (e.g. kidney, heart, brain and mesentery) primarily in the vasculature. Within the vascular wall Best‐3 was shown to be strongly expressed in both smooth muscle and endothelial cells. This was supported by PCR cloning. Specific siRNAs significantly suppressed Best‐3 expression and the I(Ca, cGMP)Cl.

Our study demonstrates the functional significance of Best‐3 protein for the cGMP‐dependent Ca2+‐activated Cl− channel in vascular tissue.

1. Matchkov et al., Circ Res 2008

2. Matchkov et al., J Gen Physiol 2004

3. Matchkov et al., Pflugers Arch 2005
LC‐MS measurement of the epoxyeicosatrienoic acid mimetic EET‐8‐ZE‐D in rat serum
Authors: Shaw Sean , Shrestha Binaya , Nithripatikom Kasem , Imig John
Published: June 2021

Epoxyeicosatrienoic acids (EET) are created by CYP450 epoxygenases and possess a wide variety of cardiovascular protective actions. Since their presence is transitory the synthesis of EET mimetics with longer lifetimes offers a potential route for cardiovascular drug development. While one such mimetic, an esterified EET analogue (EET‐8‐ZE), had no effect in SHR rats, further modification by amidation of the carboxylic group with aspartic acid (EET‐8‐ZE‐D) has been shown to reduce blood pressure in SHR rats (injected at 3mg/day; at Day 2 the MAPδ was ‐8±1.3 mmHg). From there we have developed an LC‐MS method for the detection of EET‐8‐ZE‐D in rat serum. A 200μl sample of rat serum was diluted with water to a total of 1ml then spiked with both EET‐8‐ZE‐D and deuterated‐EET standards; after adding acetic acid to a 20% concentration it underwent a chloroform/methanol liquid‐phase extraction. The organic phase was collected dried down under nitrogen, briefly resuspended in methanol then dried and suspended in 30ul acetonitrile. After reverse‐phase separation on an HPLC column (acetonitrile + 0.1% acetic acid against water + 0.1% acetic acid) the samples were run on an Agilent 1100 triple quadrupole MS, providing sufficient quantitative sensitivity to isolate 300pg. These data demonstrate that the EET‐8ZE‐D mimetic has in vivo cardiovascular activity and that EET‐8ZE‐D can be accurately measured in biological fluids.
Expression of Angiotensin Converting Enzyme 2 (ACE2) in Mice Mesangial Cells
Authors: Aragão Danielle Sanches , Andrade Maria Claudina Camargo , Casarini Dulce Elena
Published: June 2021

ACE2 is a new component of renin angiotensin system (RAS), an important physiological regulator of blood pressure, fluid and salt balance. ACE2 discovery added a new level of complexity to this system once it is able to metabolize angiotensin II, a potent vasoconstrictor, to angiotensin (1‐7), a potent vasodilator with high catalytic efficiency. Thus, this enzyme can be considered an endogenous ACE inhibitor. The relation between ACE2 and blood pressure is still controversial but this enzyme acting as a RAS negative regulator could have a vasodilator and antihypertensive potential effects. Once mesangial cells present all the components of RAS, the aim of this study was verify the expression and characterize ACE2 in immortalized mesangial cells (IMC). We purified the enzyme 5.4 fold through ion exchange chromatography with a specific activity of 40.3 nM/mg and the homogeneity was visualized by SDS‐Page. The molecular mass determined was 60 kDa. Using Western Blotting the enzyme was recognized by a specific antibody. N‐terminal sequence was determined and showed similarity with ACE2 from different species. Purified ACE2 was able to hydrolyze angiotensin II into angiotensin (1‐7), and the optimum pH and chloride concentration were 7.5 and 200 mM respectively. Enzyme activity was stable at 4º and 37ºC and it was inhibited at 1, 10 and 100μM of DX600. In conclusion, the presence ACE2 in mesangial cells may contribute to the local generation of angiotensin1‐7 and modulation of renal hemodynamic, contributing to the comprehension of the RAS. Supported by FAPESP.
Frequency response characteristics of whole body autoregulation of blood flow in rats
Authors: Stauss Harald Martin , Rarick Kevin R , Deklotz Richard J , Sheriff Don D
Published: June 2021

Previous studies from our laboratory suggested that myogenic vascular function contributes to VLF BPV. Here we tested the hypothesis that the dynamic properties of whole body autoregulation of blood flow is consistent with the frequency range of VLF BPV (0.02‐0.2 Hz) in rats.

In anesthetized rats (n=11) BPV (0.016‐0.5 Hz) was induced by computer‐regulated cardiac pacing while blood pressure and cardiac output (CO) were recorded during control conditions (NaCl, 1mL/h i.v.) and α1‐adrenergic receptor stimulation (phenylephrine, 1mg/mL/h i.v.) that has been reported to facilitate myogenic vascular function.

During control conditions, transfer function analyses between mean arterial pressure (MAP) and CO and between MAP and total vascular conductance (CO/MAP) indicated autoregulation of blood flow at 0.016 Hz, passive vascular responses between 0.033 Hz and 0.2 Hz, and baroreflex‐like responses at 0.333 Hz and 0.5 Hz. Stimulation of α1‐adrenergic receptors extended the frequency range of autoregulation of blood flow to frequencies up to 0.033 Hz.

Depending on sympathetic vascular tone, whole body autoregulation of blood flow most effectively operates at frequencies below 0.05 Hz. This frequency range is consistent with VLF BPV in rats. Baroreceptor reflex‐like mechanisms contribute to LF (0.2‐0.6 Hz) but not VLF BPV‐induced vascular responses.

Supported by the American Heart Association (0630329N)
Nitric Oxide mediating L‐glutamate cardiovascular responses in the paraventricular nucleus in unanesthetized rats
Authors: Busnardo Cristiane , Tavares Rodrigo Fiacadori , Resstel Leonardo , Crestani Carlos Cesar , Correa Fernando Morgan Aguiar
Published: June 2021

Objective

In a previous study we have reported pressor and tachycardiac responses after L‐glutamate (L‐glu) microinjection in the hypothalamic paraventricular nucleus (PVN) in unanesthetized rats. In a present study we evaluated the possible involvement of local nitric oxide (NO) mediating cardiovascular effects evoked by L‐glu injection in the PVN.

Methods

Male Wistar rats were used (250‐270g). Guide cannulas were implanted into the PVN. A catheter was introduced into the right femoral artery for blood pressure and heart rate recording.

Results

Microinjection of L‐glu in 10nmol/100 nL in the PVN caused pressor and tachycardiac responses in unanesthetized rats. Microinjections of artificial cerebrospinal fluid (ACF) in the PVN did not affect the pressor and tachycardiac responses to L‐glu microinjected in the PVN. The cardiovascular responses to L‐glu in the PVN was blocked by local pretreatment with the selective neuronal NO‐synthase (nNOS) inhibitor Nω‐Propyl‐L‐arginine (N‐Propyl, 0.04 nmol/100nL) or with the NO scavenger carboxi‐PTIO (C‐PTIO, 1nmol/100nL).

Conclusion

The present results support the idea of an involvement of PVN glutamatergic neurotransmission in cardiovascular modulation through the NO pathway.

Financial Support

FAPESP (06/52446‐9) and FAEPA.
Voluntary exercise protects against methamphetamine‐induced oxidative stress in brain microvasculature and disruption of the blood‐brain barrier
Authors: Toborek Michal , Seelbach Melissa J. , Choi Yean Jung , András Ibolya E. , Chen Lei , Hennig Bernhard , Esser Karyn A.
Published: June 2021

Moderate to vigorous exercise is a powerful means to influence health status and lower the risk of the development of chronic diseases. We hypothesize that increased antioxidant potential by endurance exercise plays a critical role in the protection against the development of cerebral toxicity associated with drug abuse. To address this hypothesis, mice were subjected to voluntary wheel running for 5 weeks. The control mice did not have access to running wheels. At the end of exercise training, mice were injected with methamphetamine (Meth, 10 mg/kg) for 24 h. Exercise markedly changed Meth‐induced alterations of cerebral blood flow and protected against Meth‐induced disruption of the blood‐brain barrier as assessed by in‐situ brain perfusion. Consistent with these results, exercise protected against Meth‐mediated disruption and localizations of tight junction protein expression (ZO‐1, occludin, and claudin‐5). Importantly, exercise markedly attenuated Meth‐induced oxidative stress in brain microvessels. It appears that protection against upregulation of NADPH oxidase may be responsible, at least in part, for these protective effects. The obtained results indicate that exercise is an important modifiable behavioral factor that can protect against Meth‐induced CNS toxicity. Supported by MH63022, MH072567, NS39254, and from the University of Kentucky Center for Muscle Biology.
Oxidative stress in the brain mediates thermogenic sympathetic activation to leptin
Authors: Rahmouni Kamal , Morgan Donald A , Mark Allyn L
Published: June 2021

Leptin acts in the brain to decrease appetite and body weight and increase sympathetic nerve activity (SNA). Leptin can increase oxidative stress as shown in endothelial cells and oxidative stress modulates SNA. Here, we examined whether oxidative stress mediates leptin‐induced increases in SNA by testing the effect of intracerebroventricular (ICV) treatment with a superoxide dismutase mimetic (Tempol, 5 μM) on the regional SNA response to intravenous leptin (1 mg/kg bw) in anesthetized Sprague‐Dawley rats. Leptin increased SNA to brown adipose tissue (BAT, 184±26%, n=6), kidney (181±29%, n=6) and hindlimb (170±19%, n=6). ICV Tempol alone had no effect on BAT (4±16%, n=8), renal (10±12%, n=8) and lumbar (14±8%, n=8) SNA. However, treatment with ICV Tempol blocked leptin‐induced sympathetic activation to BAT (‐10±19%, n=5, P<0.05 vs. leptin alone), but not to kidney (157±37%, n=5) or hindlimb (196±37%, n=5). In contrast, corticotrophin‐releasing factor‐induced increase in BAT SNA was not significantly altered by ICV treatment with Tempol (215±41%, n=6) as compared to vehicle (254±41, n=6). Our data demonstrate the importance of oxidative stress as a mediator of leptin‐induced activation of sympathetic activity to thermogenic brown adipose tissue.
Increased melanocortin activity may contribute to the reduced intake of high protein diets
Authors: Guest Dolores Doane , Barnes Meredith B , Heppner Kristy M , Beverly J Lee
Published: June 2021

Meals high in protein are associated with greater satiety and reduced intake. Increased melanocortin activity, through MC4R, in the mediobasal hypothalamus (MBH) may facilitate this reduced food intake. We tested whether the decrease in food intake observed in animals fed a high protein diet is influenced by melanocortin activity in the MBH. Melanocortin activity was altered by continuous infusion of a MC4R agonist (MTII) or antagonist (SHU 9119) into the ventricles of rats fed either a standard protein diet (C; 15:69:16) or a high protein diet (HP; 50% protein: 34% CHO: 16% fat). Male Sprague‐Dawley rats were fitted with a mini‐osmotic pump, filled with one of 3 solutions: 0.5ug/uL MTII, 0.5ug/uL SHU9119, or vehicle (0.9% sterile saline). Rats consumed 14% less high protein diet (p<0.001) during the first week of the study. Intake of rats fed the standard diet and receiving MTII was the same as vehicle‐treated rats fed the high protein diet (9.1±0.2 vs. 8.7±0.2, p>0.05). Rats fed high protein diet and receiving SHU9119 consumed the same amount of food as vehicle‐treated rats on the standard protein diet (10.5±0.6 vs. 9.9±0.1, p>0.05). These results are consistent with an increase in melanocortin activity contributing to the reduced food intake of high protein diets. DD Guest supported by APS Porter Physiology Development Award.
Net and Unidirectional Liquid Fluxes Across Porcine Bronchial Airways in Health and During Bacterial Pneumonia
Authors: Martens Chelsea J. , Schuler A. Michele , Ballard Stephen T.
Published: June 2021

The epithelium of the tracheobronchial airways is capable of both active absorption and secretion of ions and liquid to maintain adequate volumes of airway surface liquid to support mucociliary transport and prevent airway obstruction. Using cannulated, liquid‐filled bronchi from pigs, rates of liquid flux across the airway wall were measured in the presence and absence of NPPB (an anion channel blocker that inhibits liquid secretion). Net and unidirectional liquid fluxes were determined. Responses from healthy animals were compared to animals diagnosed with bacterial pneumonia (Pasteurella multocida and Streptococcus suis). Unidirectional liquid secretion in infected airways (2.35± 0.55 μl/cm2/h) was significantly (p<.05) greater than in healthy airways (0.40±0.66 μl/cm2/h). Unidirectional liquid absorption in infected airways (3.29±0.47 μl/cm2/h) was reduced relative to healthy airways (4.65±0.55 μl/cm2/h), but this difference was not significant. In healthy airways, the net liquid flux was absorptive (4.25±0.62 μl/cm2/h). In the infected airways, the net liquid flux was significantly reduced (0.93±0.43 μl/cm2/h). We conclude that the bronchial airways of animals infected with these pathogens exhibit a net reduction in absorption of airway fluid that results from both reduced unidirectional absorption and augmented unidirectional secretion. (Support: NIH 063302, CFF BALLAR07XX0)
Essential role of lactate in controlling the rapid proliferation of pulmonary microvascular endothelial cells
Authors: Parra‐Bonilla Glenda , Alvarez Diego F , Al‐Mehdi Abu‐Bakr , Stevens Troy
Published: June 2021

Pulmonary microvascular endothelial cells (PMVECs) constitutively up‐regulate glycolytic enzymes necessary to generate ATP. One of these enzymes is lactate dehydrogenase (LDH), which converts pyruvate to lactate, causing lactic acidosis in proliferating cells. Since the role of LDH and lactate in endothelial pro‐proliferation is unknown, we tested whether lactate is a key contributor of rapid PMVEC proliferation. PMVEC growth curves were generated using standard culture conditions (10% serum with 22 mM glucose) in the presence of the LDH inhibitor, oxamate (4, 8 and 16 mM). Oxamate inhibited growth, glucose consumption and lactate production in a dose‐dependent manner, suggesting that conversion of pyruvate to lactate is essential for rapid proliferation. To determine whether glycolytic flux is necessary to sustain proliferation, galactose was substituted for glucose, since galactose is an ineffective glycolysis substrate. Galactose treatment reduced cellular ATP concentrations ?2‐fold, and inhibited growth, glucose consumption and lactate production. To examine whether lactate results in glycolytic‐independent cell growth, lactate was added to galactose‐rich media. Addition of exogenous lactate restored cellular ATP concentrations and partially rescued PMVEC proliferation. Thus, LDH contributes to the rapid PMVEC proliferation by controlling the rate of pyruvate conversion to lactate.
Cell‐protective mechanisms of alpha 1 antitrypsin (A1AT) in the lung endothelium
Authors: Lockett Angelia Denise , Garrison Jana , Kamocki Krzysztof , Schweitzer Kelly , Petrache Irina
Published: June 2021

Recent studies uncovered non‐cannonical functions of A1AT that lead to cellular protection from apoptosis and inflammation, including suppression of cigarette smoke (CS)‐induced increases in TNFα. We set out to investigate the mechanisms by which A1AT antagonizes TNFα?in vivo and in primary lung endothelial cell cultures. Mice were exposed to CS for up to 6 months and total lung RNA or protein was analyzed by real‐time PCR or Western blot, respectively. Primary rat lung microvascular cells were pretreated with A1AT, followed by studies of signaling and inflammatory responses in response to TNFα. Despite a decrease in the total lung TNFα message level (21 % at 16 weeks), both IκBα and MCP1 levels increased in CS‐exposed mice (1.7 fold + sem=.075; p=.05 and 1.4 fold +sem=.029; p<0.01, respectively). A1AT treatment increased basal IκBα?and delayed the IκBα?degradation induced by TNFα (10 ng/ml) in endothelial cells by 15 min. In conclusion, although TNFα message levels are not increased in smoked animals, the increased IκBα and MCP1 expression suggest that TNFα protein is activated by cigarette smoke. At the endothelial cell level, A1AT may alter TNFα signaling by regulating IκBα degradation. Understanding the anti‐inflammatory mechanisms of A1AT is important for developing therapeutic strategies against CS‐induced emphysema.

Funded by: 1P50 HL084945