bluuebunny/crossref_metadata_2025 huggingface dataset:
L‐Arginine Enhances Nitrative Stress and Exacerbates TNF‐Alpha Toxicity in Endothelial Cells
Authors: Xia Zhengyuan , Liu Huimin , Wang Fang , Luo Tao , Xia Zhong‐yuan , Irwin Michael G , Vanhoutte Paul M
Published: June 2021

Supplementation of L‐arginine, a nitric oxide precursor, during the late phase of myocardial ischemia/reperfusion (MI/R) increases myocyte apoptosis and exacerbates myocardial injury. Apoptosis of the endothelial cells precedes that of the cardiomyocytes during MI/R, and this is associated with increased tumor necrosis factor‐alpha (TNF) production. We postulated that L‐arginine may exacerbate TNF induced endothelial cell apoptosis by enhancing nitrative stress. Cultured human umbilical vein endothelial cells were studied under control conditions or treated with TNF alone or in the presence of L‐arginine the non‐selective nitric oxide synthase inhibitor N (omega)‐nitro‐L‐arginine (L‐NNA), propofol (scavenger of peroxynitrite), or L‐arginine plus propofol, respectively, for 24 hours. TNF increased endothelial cell apoptosis, increased the intracellular superoxide production and the protein expression of nitrotyrosine, a footprint of peroxynitrite formation and an index of nitrative stress. L‐arginine exacerbated all these changes while L‐NNA or propofol attenuated them. Thus, under pathological conditions associated with increased TNF production, L‐arginine supplementation may further exacerbate TNF cellular toxicity by enhancing nitrative stress.

Supported by a Society of Cardiovascular Anesthesiologists Research Starter Grant (to ZX).
Inhibitory effect of betulinic acid on TNF‐α‐induced adhesion molecule and its related signaling
Authors: Yoon Jung Joo , Lee Yun Jung , Kang Dae Gill , Kim Jin Sook , Lee Ho Sub
Published: June 2021

Vascular inflammation is an important event in the development of vascular diseases including atherosclerosis. In the present study, we investigated whether betulinic acid (BA) inhibits vascular inflammation process induced by tumor necrosis factor‐alpha (TNF‐α) in human umbilical vein endothelial cells (HUVEC). Pretreatment with BA was significantly blocked TNF‐α‐induced expression levels of cell adhesion molecules such as vascular cell adhesion molecule‐1 (VCAM‐1), intracellular adhesion molecule‐1 (ICAM‐1), and endothelial cell selectin (E‐selectin). Gelatin zymography results showed that TNF‐α‐induced metalloproteinase (MMP)‐2 and MMP‐9 activities were prevented by pretreatment with BA. Real time qRT‐PCR data also showed that BA suppressed TNF‐α‐induced increase in VCAM‐1, ICAM‐1, and E‐selectin mRNA expression levels. In addition, BA inhibited the TNF‐α‐induced intracellular reactive oxygen species (ROS) production. Furthermore, pretreatment with BA significantly inhibited the translocation to nucleus and transcriptional activity of NF‐κB increased by TNF‐α. In conclusion, the present data suggested that BA could suppress TNF‐α‐induced vascular inflammatory process, due to the inhibition of ROS and NF‐κB activation in HUVEC.
Neurobehavioral Effects of Gestational and Lactational Exposures to Manganese (Mn) and Lead (Pb)
Authors: Phattanarudee Siripan , Böhlke Mark , Chang Julie , Maher Timothy J
Published: June 2021

Gestational (G) and lactational (L) exposures to Mn and Pb can produce neurotoxicity, and possibly result in impaired learning, hyperactivity and attention deficits. We studied the behavioral effect in the offspring of exposed rat dams via their drinking water (MnCl2 1.25, 5 and 10 mg/ml and Pb acetate 25, 250, 2500 µg/ml) during G&L. Morris water maze (MWM) was used for testing learning and memory at PND 35 and elevated plus maze (EPM) was used for testing impulsivity and hyperactivity at PND 56. Males exposed to Mn showed the memory impairment in the MWM probe trial. Females exposed to Mn (5&10) also had the tended to display impaired memory, however not statistically significant. Only female Mn (1.25&5) displayed hyperactivity (higher velocity), while male rats exposed to Mn were similar to controls. Males exposed to Pb (25&250) showed memory impairment. For female rats, the same tendency of impairment was observed from all doses of Pb. Male rats exposed to Pb at any level did not show impulsivity or hyperactivity compared to the controls in the EPM. However, female rats exposed to Pb (250) showed increased impulsivity and hyperactivity. This study shows that G&L exposure to Mn and Pb produces some subtle neurobehavioral effects of the offspring and that these were partly gender specific. Supported by NIH grant ES‐03‐004.
Opioid‐induced preconditioning is dependent on caveolin‐3 expression
Authors: Tsutsumi Yasuo M. , Horikawa Yousuke T. , Roth David M. , Patel Hemal H.
Published: June 2021

Caveolae are flask‐like invaginations of the sarcolemmal membrane. Recently, we have shown that caveolar structural proteins, caveolins, are essential in cardiac protection and that cardiac specific overexpression of caveolin‐3 (Cav‐3 OE) results in innate cardiac protection. Additionally, we identified that opioid‐induced preconditioning (OPC) is absent when caveolae are disrupted in vitro. However, the impact of caveolin‐3 and caveolae on OPC in vivo has not been investigated. 8‐12 week old Cav‐3 OE, caveolin‐3 knockout mice (Cav‐3 KO), and control mice were exposed to 30 min ischemia/2 hr reperfusion (I/R). Cav‐3 OE and control mice were treated with naloxone, a non‐selective opioid antagonist, 10 min prior to I/R to determine whether basal protection was abolished. Cav‐3 KO and control mice were treated with SNC, a delta‐selective opioid agonist 15 min prior to I/R to induce OPC. Cav‐3 OE mice displayed endogenous cardiac protection from I/R when compared to control mice (20±2% vs. 41±2%, n=8, respectively). This endogenous protection in Cav‐3 OE mice was abolished with naloxone pretreatment (41±2%). Conversely, control mice were protected in the presence of SNC (28±1%), although no protection was observed in Cav‐3 KO mice (38±5%). These results suggest that OPC is dependent on caveolin‐3 expression and endogenous protection induced in Cav‐3 OE mice may be opioid dependent.
Gardenia jasminoides inhibits TNF‐α‐induced adhesion molecule and NF‐κB expression
Authors: Hwang Sun Mi , Lee Yun Jung , Kang Dae Gill , Lee Ho Sub
Published: June 2021

Inflammatory mediators such as TNF‐α and interleukin‐8 (IL‐8), which can enhance binding of low‐density lipoprotein (LDL) to endothelium and upregulate expressions of leukocyte adhesion molecules on endothelium during atherogenesis. The present study was designed to examine the effect of ethanol extract of Gardenia jasminoides (EGJ) on vascular inflammation response in primary cultured human umbilical vein endothelial cells (HUVECs). We found that TNF‐α‐induced intracellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), and endothelial‐selectin (E‐selectin) protein expressions and mRNA levels were inhibited by EGJ in HUVECs. A further analysis indicated that EGJ attenuated TNF‐α‐induced IκB phosphorylation, NF‐κB p65 expression, suggesting that EGJ primarily affects TNF‐α‐induced NF‐κB signaling pathway. In a functional study, EGJ dose‐dependently attenuated monocyte adhesion to endothelial monolayer. Taken together, we provided here the first evidence showing that EGJ is able to inhibit TNF‐α‐induced NF‐κB activation, adhesion molecule expression, and monocyte‐endothelial interaction, suggesting an anti‐inflammatory role of EGJ and possibly explained EGJ can prevent vascular diseases.
Dual modulation of urinary bladder function and urine production by EP3 receptors in the conscious rat
Authors: Thorneloe Kevin , Jugus Malcolm , Jaworski Jon‐Paul , Patra Phani , Jin Jian , Morrow Dwight , Laping Nicholas , Edwards Richard
Published: June 2021

COX inhibitors function to reduce levels of PGE2 and are broadly efficacious in overactive bladder models. Previously we have shown that KO mice lacking the PGE2 receptor, EP3, demonstrate an enhanced bladder capacity and resistance to PGE2‐induced bladder overactivity. We therefore investigated a regulation of urinary bladder function in conscious rats via pharmacological modulation of the EP3 receptor. In conscious, continuous‐filling cystometry of spontaneously hypertensive rats the EP3 agonist GR63799X reduced bladder capacity. In contrast, EP3 antagonists CM9 and DG041 significantly enhanced bladder capacity. The responses to GR63799X were inhibited by CM9 and DG041 in vivo supporting the activity of these pharmacological agents at the EP3 receptor. In addition to the effect on bladder capacity GR63799X increased endogenous urine production. Intra‐arterial infusion of saline mimicked the enhancement of urine production observed with GR63799X and the response was inhibited by CM9. These pharmacological data confirm the phenotype reported in EP3 receptor KO mice and further support the EP3 receptor as a modulator of urinary bladder activity. In addition, this study suggests a role for EP3 activity in the regulation of urine production.
Regulation of Carcinoma Invasion by ADAMs and MMPs
Authors: Toker Alex
Published: June 2021

The metastatic dissemination of carcinoma cells is regulated by numerous genetic and epigenetic events. The acquisition of a mesenchymal phenotype is concomitant with a propensity of cells to undergo invasive migration. Studies have revealed that matrix proteases operate at the tumor‐stromal boundary. Two families of matrix proteases that are crucial for invasive migration are the ADAMs (A Disintegrin and Metalloproteinase) and MMPs (Matrix Metalloproteinases), which are secreted by either tumor cells or by the stroma. ADAM9 is upregulated in a large number of breast tumors and cell lines. We have shown that ADAM9 is highly expressed in tumor cell lines, and expression and shRNA studies show that ADAM9 promotes invasive migration of breast cancer cells. An analysis of the functional mechanisms by which ADAM9 promotes invasive migration will be presented. We have also found that the transcriptional factor FOXO3a induces the expression of MMP‐13. In turn, this promotes the invasive migration of breast cancer cells. In vivo studies and pathological examination of tumors show that FOXO3a and MMP‐13 are highly expressed in breast tumors. Thus, ADAMs and MMPs are crucial for the efficient dissemination of highly aggressive carcinoma cells.
The delayed release of PGE2 mediated by PAR1 involves slowly developing production of arachidonic acid in rat gastric mucosal epithelial cells
Authors: Maeda Yuma , Sekiguchi Fumiko , Ohi Ai , Takaoka Kaori , Nishikawa Hiroyuki , Kawabata Atsufumi
Published: June 2021

We previously showed that activation of proteinase‐activated receptor‐1 (PAR1) caused delayed (18 h later) PGE2 release in rat gastric mucosal epithelial RGM1 cells, although the maximal up‐regulation of COX‐2 protein was observed much earlier (6 h later). In this study, we analyzed mechanisms for the delay of PGE2 release caused by PAR1 stimulation in RGM1 cells. Stimulation with a PAR1‐activating peptide, TFLLR‐NH2 (TF), in combination with arachidonic acid (AA) or linoleic acid (LA) induced rapid and synergistic increase in PGE2 release within 3 h. CP‐24879, a Δ5/Δ6‐desaturase inhibitor, suppressed the PGE2 release induced by TF plus LA, but not by TF plus AA or TF alone, at 18 h. The TF‐induced delayed PGE2 release was suppressed by combined application of inhibitors of cPLA2, iPLA2 and sPLA2, but not by each of them. Protein levels of microsomal PGE synthase (mPGES)‐1, mPGES‐2 and cytosolic PGES were unchanged by stimulation with TF. These data suggest that the delay of PAR1‐triggered PGE2 release is due to slowly developing AA production in RGM1 cells, and that three isozymes of PLA2 may complementarily contribute to the AA production.
Modulation of butyrate uptake by Caco‐2 cells
Authors: Gonçalves Pedro Filipe Fernandes , Araújo João Ricardo , Pinho Maria João , Martel Fátima
Published: June 2021

The aim of this study was to investigate the putative influence of some pharmacological agents and drugs of abuse upon the apical uptake of butyrate (BT) by Caco‐2 cells. Apical uptake of 14C‐BT by Caco‐2 cells seems to involve MCT1‐mediated transport (it was time‐, concentration‐, pH‐ and energy‐dependent, Na+‐independent, Cl− ‐dependent, inhibited by several BT structural analogues, insensitive to anion exchange inhibitors and inhibited by monocarboxylate transport (MCT) inhibitors). Acutely, uptake of 14C‐BT (10 µM) was reduced by acetaldehyde (ACA), acetylsalicylic acid (ASA), indomethacin, caffeine and theophylline, and increased by MDMA. Chronically, it was increased by caffeine and decreased by tetrahydrocannabinol (THC) and MDMA; qRT‐PCR analysis showed that these three compounds decreased the mRNA levels of MCT1. Acutely, ACA, indomethacin and MDMA reduced the uptake of 14C‐BT (20 mM), and ASA increased it. Chronically, none of the compounds affected uptake. ACA, indomethacin and propionate seem to be competitive inhibitors of 14C‐BT uptake. ASA simultaneously increased the Km and the Vmax of 14C‐BT uptake. In conclusion, MCT1‐mediated transport of 14C‐BT in Caco‐2 cells is modulated by either acute or chronic exposure to some pharmacological agents and drugs of abuse (ACA, ASA, indomethacin, caffeine, theophylline and the drugs of abuse THC and MDMA).

Supported by FCT (PTDC/SAU‐FCF/67805/2006)
The role of conserved transmembrane domain residues in the activation of the dopamine D2 receptor
Authors: Tambe Mitali , Cumbay Medhane
Published: June 2021

The interaction between two highly conserved amino acid residues in second (TM2) and seventh (TM7) transmembrane domains (aspartate and asparagine respectively) of GPCRs plays an important role in receptor activation and coupling specificity to different signal transduction pathways. Although single mutations of each conserved residue can selectively alter receptor signaling, reciprocal mutations in TM2 and TM7 appear to partially restore function in a receptor dependent manner. For example, reciprocal mutations in alpha2a‐adrenergic and 5HT2a serotonin receptors restores receptor function, but reciprocal mutation in the CB1cannabinoid receptor does not. We have characterized the role of the two conserved residues in the activation and signaling of the D2 dopamine receptor. We report that the D80N mutation in TM2 of the D2 receptor ablates receptor‐dependent activation of Kir 3.0 channels and significantly blunts receptor‐dependent inhibition of cyclic AMP accumulation. In contrast to the corresponding mutant (D164N) in the CB1 receptor, the D80N mutation also ablated receptor‐dependent phosphorylation of ERK1/2. We also report that the reciprocal mutant in D2 receptor restores receptor‐dependent phosphorylation of ERK1/2 and activation of Kir3.0 channels indicating a receptor specific effect of the conserved residues. This work is supported by NIP grant from the AACP.
Fast dilatory responses to potassium in arterioles of the rat gastrocnemius muscle (G): impact of branch order
Authors: Roseguini Bruno Tesini , Thorne Pam , Davis Michael J , Laughlin M. Harold
Published: June 2021

Rapid dilatory responses to muscle contractions are nonuniform within the arteriolar tree, starting at distal segments and spreading upstream. Potassium (K+) is a putative vasodilator involved in the immediate exercise hyperemia, but it is not known if a longitudinal gradient in the responsiveness to K+ exists within the arteriolar network. We tested the hypothesis that smaller arterioles of the G arteriolar network respond faster and to a greater extent to application of K+ than proximal segments. First‐order (1A's) and third‐order (3A's) arterioles (red portion of G) were isolated, cannulated and pressurized at 85 and 53 cmH2O, respectively. Luminal diameter was monitored continuously (30Hz) by a tracking system during: 1) exposure to cumulative doses of potassium chloride (KCl) (2.5mM to 15mM) and 2) application of a brief pulse of 30mM KCl using a micropipette positioned in the vicinity of the arteriolar wall. Both arteriolar orders dilated similarly in response to increasing doses of KCl, reaching peak dilation at the 12.5 mM dose (1A's: 33±8% vs. 3A's: 32±15%). The time delay to the onset of vasodilation (TD) (1A's: 3.3±1.9s vs. 3A's: 2.3±1.2s), time‐to‐peak (1A's:11.6±7.1s vs. 3A's: 6.3±2.3s) and magnitude of dilation (1A's:11±7% vs. 3A's:13±7%) in response to local application of KCl were not different between branch orders. These findings show that 1A's and 3A's have similar sensitivity to potassium. Additionally, the overall TD to vasodilation (~2.5s) supports the role of potassium as a mediator of the immediate exercise hyperemia. Support: HL 36088 and ACSM Foundation Research Grant.
Nitric Oxide (NO) Bioavailability Underlies Muscle Microvascular O2 Delivery/Utilization Imbalance in Chronic Heart Failure (CHF) Rats
Authors: Hirai Daniel M , Copp Steven W , Ferreira Leonardo F , Musch Timothy I , Poole David C
Published: June 2021

NO bioavailability has been implicated in the muscle microvascular dysfunction of CHF at rest and during contractions.

PURPOSE

To test the hypothesis that differences in the O2 delivery/utilization balance, which sets the microvascular O2 partial pressure (PO2mv), between control (C) and severe CHF rats following the onset of contractions are due substantially to NO reduction.

METHODS

PO2mv was measured via phosphorescence quenching in the spinotrapezius muscle of 6 C and 4 CHF rats (male Sprague‐Dawley, 7‐10 wk post‐myocardial infarction, LVEDP 35+2 mmHg). Separate electrically‐induced contraction bouts (1 Hz) were performed with superfusion of SNP (300 μmol), Krebs‐Henseleit buffer (KH), and L‐NAME (1.5 mmol). The area under the PO2mv profile (PO2AREA) was calculated from 0‐60 s of each bout (expressed below as mmHg·s/min).

RESULTS

For KH, C had a greater (P < 0.05) PO2AREA (1042 ± 62) than CHF (721 ± 141). SNP increased PO2AREA (C: 1371 ± 59, CHF: 1263 ± 97, P > 0.05) whereas L‐NAME reduced PO2AREA (C: 613 ± 74, CHF: 416 ± 70, P > 0.05) to equivalent values in C and CHF rats.

CONCLUSIONS

This demonstrates, for the first time, that the impaired muscle microvascular O2 delivery/utilization balance in CHF is eliminated by increasing (SNP) or removing (L‐NAME) NO bioavailability suggesting that the dysfunctional CHF PO2mv profile results principally from endogenous NO alterations.

AHA, Heartland Affiliate 0750090Z
Causes for spatial heterogeneity of perfusion and function
Authors: Pries Axel R , Reglin Bettina , Secomb Timothy W
Published: June 2021

In many tissues, including the heart, perfusion and metabolic activity are heterogeneous and spatially correlated. To investigate the underlying mechanisms and cause/effect relations, a mathematical simulation model based on experimental data for microvascular networks was used. Results show that unavoidable geometric heterogeneities of vascular networks cause heterogeneity of blood flow and oxygen levels which cannot be eliminated by increasing metabolic sensitivity of diameter adaptation. Additional adaptation of oxygen demand to local oxygen availability allows increased oxygen extraction and functional capacity. It also establishes a correlation between local oxygen demand and flow rate, as observed experimentally. These findings suggest that stochastic processes of angiogenesis and not pre‐existing heterogeneities of local metabolic demand underlie the observed spatial heterogeneity of perfusion and function.
Protection from Diabetes‐Associated Apoptosis with Heat Shock Protein 27 Overexpression
Authors: Williamson Courtney L , Dabkowski Erinne R , Brock Robert W , Frisbee Jefferson C , Hollander John M
Published: June 2021

Diabetic cardiomyopathy is associated with cardiac contractile abnormalities which may result from and enhanced apoptotic susceptibility. The small heat shock protein 27 (hsp27) contributes to preservation of cytoskeletal structure which may impact the apoptotic program. The goal of this study was to determine whether hsp27 overexpression influences diabetes‐induced apoptosis. Transgenic mice overexpressing hsp27 and littermate controls were made diabetic by streptozotocin injection and sacrificed seven weeks following diabetic onset. Hsp27 overexpression preserved cardiac contractile function in the diabetic heart as indicated by enhanced rates of relaxation and contraction, as well as developed pressures (P<0.05 for all three). Caspase activity was decreased in diabetic hearts overexpressing hsp27 as compared to diabetic littermate controls (P<0.05). Cytochrome c and apoptosis‐inducing factor content in the cytosolic fraction was decreased in diabetic hearts overexpressing hsp27 as compared to diabetic littermate controls (P<0.05 for both). There was an increased association of hsp27 and alpha‐actin in diabetic hearts overexpressing hsp27 as compared to diabetic littermate controls. These results suggest that hsp27 overexpression attenuates cardiac contractile abnormalities resulting from diabetic insult, which is associated with a decrease in apoptosis susceptibility.
Alterations in energy substrate utilization associated with high dietary fat in the infarcted rat heart
Authors: Berthiaume Jessica Marie , Chen Xiaoqin , McElfresh Tracy A , Chandler Margaret P
Published: June 2021

Lipid accumulation in non‐adipose tissues, such as the heart, causes toxicity suggesting excess lipid is pathological. However, our work suggests high dietary fat subsequent to myocardial infarct results in less contractile dysfunction & increased β‐oxidation enzyme activity than a standard diet. This study aimed to define the impact of high fat (SAT) on cardiac energy metabolism after infarction. Rats underwent coronary ligation (HF) or sham (SH) surgery & were fed SAT (60% kcal fat) or normal chow (NC) for 8 weeks with echocardiographic evaluation. Hearts were perfused in working mode with 14C glucose and 3H oleate for metabolic analysis in coronary effluent. Hearts were also monitored for output, heart rate, & oxygen uptake. Results show function was similar across groups, but oxygen consumption decreased 20% with HF. Glucose oxidation was increased in HF, but increased less in HF‐SAT. Fatty acid oxidation was increased in both SAT groups. Lower oxygen consumption & increased glucose utilization suggests less aerobic metabolism, in agreement with literature on HF. Lower elevations of glucose oxidation in the HF‐SAT group suggests altered metabolism is less pronounced with high fat, further supported by the elevated rates of fatty acid oxidation in the HF‐SAT group. Results suggest high fat is protective of function by maintaining fatty acid metabolism in the heart. (Supported by NHBLI HL‐081857 & AHA‐0535361N).
Effect of Resistance Exercise with Blood Flow Restriction on Muscle Protein Synthesis and mTOR Signaling in Older Men
Authors: Fry Christopher S , Glynn Erin L , Drummond Micah J , Timmerman Kyle L , Fujita Satoshi , Abe Takashi , Sato Yoshiaki , Dhanani Shaheen , Volpi Elena , Rasmussen Blake B
Published: June 2021

Recent studies have shown low‐intensity (20~50% of 1‐repetition maximum) resistance training combined with a moderate reduction of vascular flow to working muscles produces similar increases in hypertrophy and strength compared to traditional high‐intensity training. Seven male subjects (age > 60 years) performed low intensity resistance exercise on separate days with (BFR n=7) or without (Control n=7) a blood flow restriction cuff placed proximally on each leg. Blood samples were obtained throughout the study and muscle biopsies were taken at baseline, immediately, 1 and 3hr post‐exercise. Mammalian target of rapamycin (mTOR) signaling was assessed using immunoblotting methods. mTOR phosphorylation increased post‐exercise in both groups. S6K1 phosphorylation increased at 3hr post‐exercise in the BFR group only. rpS6 phosphorylation was higher 1 and 3hr post‐exercise in the BFR group and higher at only 1 hr post‐exercise in the Control group. The increase from baseline for muscle protein synthesis during post‐exercise recovery was ~80% in the BFR group and only ~10% in the Control group. We conclude that blood flow restriction during low intensity resistance exercise appears to enhance muscle protein synthesis and mTOR signaling in older men which may be an effective strategy to counteract sarcopenia.

Supported by NIH/NIAMS RO1 AR049877, NIH/NIA P30AG024832, UTMB Center for Rehabilitation Sciences
Lifelong wheel running exercise and 8% caloric restriction attenuate Caspase‐independent apoptosis via downregulation of EndoG in the Aging Fischer‐344 rat plantaris
Authors: Kim Jong‐Hee , Lawler John M
Published: June 2021

Aging is characterized by a progressive loss of muscle mass and muscle fiber atrophy or ¡°sarcopenia.¡ ±Although the molecular mechanisms responsible for sarcopenia have not been completely understood, recent literature indicates that mitochondrial dysfunction and apoptosis play critical roles in sarcopenia. Regular exercise and caloric restriction provide significant protection against progressive sarcopenia. We tested the hypothesis that lifelong wheel running exercise combined with mild (8%) caloric restriction (8%CR) would attenuate caspase‐independent apoptosis via downregulation of endonuclease G (EndoG) and apoptosis inducing factor (AIF) in the aging plantaris muscle. Male Fischer‐344 rats were divided into 4 groups at 11 weeks of age: ad libitum feeding until 6 mo. old (6AL, n=12); fed ad libitum until 24 mo. old (24AL, n=11); 8%CR to 24 mo of age (24CR, n=12); wheel running to 24 mo. + 8%CR (24ExCR, n=12). EndoG protein level was increased with age in both nucleosome fraction (+117%) and soluble fraction (+15%). 8%CR and wheel running exercise ameliorated age‐induced increases in EndoG in nucleosome fraction (‐37%). AIF protein level was not altered by age in both soluble and nucleosome fraction. These data indicate that lifelong wheel running and 8%CR attenuated age‐related increased in caspase‐independent apoptosis through EndoG translocation into nucleus.
Comparison of advanced age‐related denervation and fiber type grouping in two different strains of homozygous Sod1 knockout mice
Authors: Kostrominova Tatiana Y
Published: June 2021

Cu/Zn superoxide dismutase (SOD1) is an enzyme critically important for superoxide free radical elimination. In this study age‐matched wild type (Sod1+/+) and heterozygous (Sod1−/+) littermates were compared to ~9‐12 month old SOD1 homozygous knockout (Sod1−/−) mice from two different strains: Jackson Laboratory (JL; C57Bl6/129SvEv background) and Dr. C.J. Epstein Laboratory (EL; C57Bl6 background). Sod1−/− mice from both JL and EL had smaller body and skeletal muscle masses. Following staining for succinate dehydrogenase, grouping of positive fibers was observed in muscles of EL Sod1−/− mice (Kostrominova et al., 2007) and JL Sod1−/− mice, but not in age matched Sod1−/+ mice. Total body weight and skeletal muscle mass of 45 day old Sod1−/− mice from JL were not different from age‐matched Sod1−/+ mice but their muscles showed distinct fiber type grouping. EDL, PLT and TBA muscles from ~9‐12 month old Sod1−/− mice from EL did not display denervation and GTN muscle displayed a very small degree of denervation (Kostrominova et al., 2007). By contrast, age‐matched Sod1−/− mice from JL displayed pronounced denervation of GTN muscle as well as denervation of other hind limb muscles. I conclude that genetic background can have a significant influence on how the lack of SOD1 enzymatic activity effects the rate of age‐related skeletal muscle denervation and fiber type grouping.
Human genomic microarrays reveal novel processes that may initiate skeletal muscle regeneration in older adults
Authors: Thalacker‐Mercer Anna Elizabeth , Dell'Italia Louis J. , Mayhew David L. , Cui Xiangqin , Bamman Marcas M.
Published: June 2021

Aging skeletal muscle demonstrates an impaired and poorly understood capacity for regeneration and growth when provided the same stimuli as young. To better understand the impact of aging and to identify novel responses for targeted studies, we assessed changes in gene expression (GE) of 8 old vs. 8 young humans pre and 24‐h post unaccustomed high‐intensity resistance loading (RL). Within each age group, RL‐mediated changes in GE were determined using a paired t‐test (significance determined at P<0.001). Ingenuity Pathways Analysis (IPA) was used to identify functional networks. Among old, translated protein products of transcripts in the highest scoring network play a role in skeletal and muscular development (DYRK1B, MAP2K3, MYBPH, NRP1, SMARCA4, JUP, PLXNA1); cell growth and proliferation (TCP1, CCT5, FADS3, COL4A3); and inflammation (Heatshock proteins, NFkB, p38MAPK, ERK, MAP3K5, ZFG36). Two transcripts on this network (NFkB and HSP70) were differentially expressed in young post RL. Although acute inflammation is thought to play an important role in initiating early steps of RL‐mediated myogenesis, a heightened inflammatory response (mRNA level) in old may blunt transcriptional changes which support muscle development and growth. Targeted studies in our lab will determine whether these novel transcriptome changes, unique among old, are responsible for age‐related muscle regeneration impairment. Supported by: NIA 5R01 AG017896, 5T32 DK062710‐01A1, GCRC M01 RR00032
Effect of exercise dose on fat oxidation capacity in postmenopausal women
Authors: Johannsen Neil Michael , Edwards W. Brent , Church Timothy S. , Blair Steven N , Earnest Conrad P.
Published: June 2021

The capacity to oxidize fat may play an important role in health and disease. However, the effect of exercise dose on maximal fat oxidizing capacity remains unexplored. To investigate this relationship, we utilized a sub‐cohort of women (n = 260) from the DREW study (n = 464) who met stringent VO2max criteria before and after 6 months of exercise. Women were randomly assigned to doses of exercise (kcal·kg body weight−1·week−1; KKW) at 50% VO2max; control (CON; n = 54), 4 KKW (n = 82), 8 KKW (n = 62), and 12 KKW (n = 62). Maximal fat oxidation (MFO; g/min) and intensity eliciting MFO (FatMax) were calculated from VO2max data. Overall, our current sub‐cohort had similar improvements in VO2max to those previously published by our group (trend P < 0.001). Mean (±SD) baseline MFO and relative FatMax (%) were 0.20 ± 0.06 g/min and 51.3 ± 6.3%, respectively. Neither MFO (P = 0.93) nor FatMax (%; P = 0.51) increased with training; however, improvements in absolute FatMax (mL·kg−1·min−1) were greater after training at 8KKW (0.7 ± 1.2) and 12KKW (1.0 ± 1.1) compared with CON (0.0 ± 1.4) and 4KKW (0.1 ± 1.1; P < 0.001). In conclusion, exercise at 50% VO2max for 6 months does not significantly improve MFO or relative FatMax. The data suggest that 50% VO2max may not be adequate to improve MFO. Absolute FatMax data indicate that the workload during training must be constantly increased at higher doses of exercise in order to optimize fat oxidation. This research was funded by grant HL66262 from the National Institutes of Health.
Vascular reactivity in femoral arteries of aged rats following resistance exercise training
Authors: McFaden Jennifer B. , Araj Christina A. , Richards Margaret A. , Harris M. Brennan
Published: June 2021

Previous studies have demonstrated an increase in arterial stiffness and endothelial dysfunction in aging arteries. Although it is well known that resistance training counteracts losses of muscular strength with age and subsequent loss in quality of life, few studies have examined the effects of resistance training on vascular function. Young (9 mo) and aged (24 mo) male, Fisher 344 rats were divided into four groups: Young Sedentary (YS, n=10), Young Trained (YT, n=10), Aged Sedentary (AS, n=9), and Aged Trained (AT, n=9). Resistance training consisted of climbing a 1 m wire ladder, at an 85o angle, 3d/wk for 7 weeks with increasing weight added to the tail. Vascular reactivity was examined by constructing dose response curves to phenylephrine (PE, 10−7‐10−4 M), sodium nitroprusside (SNP, 10−8‐10−5 M), and acetylcholine (ACh, 10−7‐3x10−5 M). Vasocontrictor responses to PE were significantly (P<0.05) greater in Aged versus Young. Vasodilator responses to both SNP and ACh were significantly impaired in AS but were improved in AT. In conclusion, these data suggest that resistance exercise training results in a shift in the vasoreactivity of aged animals towards that of younger animals which includes both a blunted vasoconstrictor response and enhanced vasorelaxation. The mechanisms underlying this shift remain to be determined. Supported by NIH R15 HL082649‐01 (MBH) and the Borgenicht Program for Aging Studies and Exercise Science (MBH).
SYMPATHETIC ACTIVITY TO DISTINCT TARGETS FOLLOWING STIMULATION OF THE ROSTRAL VENTROLATERAL MEDULLA
Authors: Mueller Patrick J , Scislo Tadeusz J
Published: June 2021

Baroreflex mediated sympathoexcitation is associated with greater activation of pre‐ganglionic adrenal nerve activity (pre‐ASNA) compared to renal (RSNA) or lumbar (LSNA) sympathetic nerve activity (Am J Physiol 275: R995‐R1002, 1998). We hypothesized that a similar pattern of differential activation may also occur with direct activation of the rostral ventrolateral medulla (RVLM), the primary brain region responsible for resting and reflex mediated changes in SNA. Therefore, in urethane/chloralose anesthetized rats (n=3), we performed unilateral microinjections of glutamate (30‐90 nl, 1‐100 mM) into the RVLM while simultaneously recording mean arterial pressure (MAP), pre‐ASNA, RSNA, and LSNA in the same animals. Glutamate increased MAP, and differentially increased SNA in all three nerves with a trend toward pre‐ASNA > RSNA > LSNA. Activation of the RVLM with different volumes of glutamate (10 mM, 30‐90nl) also produced different patterns of regional sympathoactivation and these varied across animals. These experiments suggest that increased SNA to distinct targets varies depending on the manner in which the RVLM is activated. These data are also consistent with previous reports that suggest viscerotopic organization of the RVLM. (AHA‐0650161Z; NIH HL‐6714)
5α‐reductase mRNA is increased in the rostral ventrolateral medulla (RVLM) of term pregnant rats
Authors: Heesch Cheryl M. , Burcks Shannon
Published: June 2021

In term pregnant rats, baseline GABAergic inhibition of the RVLM is increased, while baseline GABAergic inhibition of the hypothalamic paraventricular nucleus (PVN) is decreased. The major metabolite of progesterone, 3α‐hydroxy‐dihydroprogesterone (3α‐OH‐DHP), is a potent endogenous positive modulator of GABAA receptors and CNS levels of this neurosteroid are increased in pregnancy. Local regulation of the enzymes responsible for formation of 3α‐OH‐DHP, 5α‐reductase (5α‐RDA) and 3α‐hydroxysteroid dehydrogenase (3α‐HSD), is one factor which determines brain region specific actions of 3α‐OH‐DHP. Expression of mRNA for 5α‐RDA and 3α‐HSD (reference gene = MAPK) was determined in tissue punches of the RVLM and PVN from nonpregnant (NP) and term pregnant (P) rats. In the PVN, pregnancy did not change relative expression of 5α‐RDA [NP(7)= 1.0 ±0.23; P(7)= 0.64± 0.15] or 3α‐HSD [NP(5)= 1.0 ±0.38; P(6)= 0.84± 0.23]. In the RVLM, expression of 3α‐HSD [NP(5)= 1.0 ±0.54; P(6)= 0.67± 0.44] was not different between NP and P rats. However, there was a strong trend for increased expression of 5α‐RDA in P (5) [2.44± 0.89] compared to NP(5) [1.0 ±0.28] rats. Although preliminary, these data suggest that increased expression of 5α‐RDA in the RVLM of pregnant rats, could result in increased formation of the major metabolite of progesterone and potential positive modulation of GABAA receptors. (NIH HL 36245)
Sympathoexcitatory Response to Exogenous Angiotensin II in the Rostral Ventrolateral Medulla Depends on Local Iontotropic Glutamate Receptors
Authors: Adams Julye M , Stocker Sean D
Published: June 2021

Glutamate and angiotensin II (AngII) regulate the excitability of sympathoexcitatory neurons in the rostral ventrolateral medulla (RVLM). However, limited evidence is available regarding whether these two neurotransmitters interact within the RVLM to regulate sympathetic nerve activity (SNA) and arterial blood pressure (ABP). Therefore, the present study examined whether blockade of either iontotropic glutamate or AngII‐type 1 receptors modulated the sympathoexcitatory response to exogenous AngII or glutamate, respectively. In urethane‐chloralose anesthetized rats, unilateral injection (60nL) of the AT1R antagonist losartan (1nmol, n=3) into the RVLM did not affect the increase in renal SNA (114±12 vs 97±14%) and mean ABP (27±2 vs 24±1 mmHg) to 1nmol glutamate. Losartan did eliminate the increase in renal SNA (50±6 vs 0±0%) and ABP (20±2 vs 0±0 mmHg) evoked by 60pmol AngII. In marked contrast, unilateral injection of the iontotropic glutamate receptor antagonist kynurenic acid (3nmol, n=6) into the RVLM eliminated the AngII‐evoked increase in renal SNA (50±6 vs 1±1%) and mean ABP (24±1 vs 0±1mmHg) but not the sympathoexictatory response to 1nmol carbachol (renal SNA: 45±2%; mean ABP: 32±2mmHg, n=3). These findings suggest that iontotropic glutamate receptors mediate the sympathoexcitatory response to exogenously applied AngII in the RVLM. Supported by NIH HL090826, AHA 0630202N, AHA0815372D
Ras‐activated caspase 3‐dependent apoptosis through MAPK and p53 in RVLM increases sympathetic nerve activity in SHRSP
Authors: Kishi Takuya , Hirooka Yoshitaka , Ito Koji , Araki Shuichiro , Konno Satomi , Sunagawa Kenji
Published: June 2021

Background

The small G protein Ras causes caspase‐3 dependent apoptosis through mitogen‐activated protein kinase (MAPK), extracellular signal‐regulated kinase (ERK) and p53. Angiotensin II administered into the rostral ventrolateral medulla (RVLM) induces pressor response through the activation of p38 MAPK and ERK. We hypothesized that Ras‐activated caspase‐3 through MAPK/ERK/p53 in RVLM increases SNA in stroke‐prone spontaneously hypertensive rats (SHRSP),

Methods and Results

Activity of Ras, MAPK, ERK and caspase‐3 and expression of p53 in RVLM were significantly higher in SHRSP (14‐16 weeks old) than in age‐matched Wistar‐Kyoto (WKY) rats. In SHRSP, intracerebroventricular (ICV) infusion of S‐farnesylthiosalicylic acid, a Ras inhibitor, for 14 days significantly reduced blood pressure (BP), heart rate (HR) (‐28±4 mmHg and ‐44±6 bpm, n=5, p<0.01) and SNA (‐28%, n=5, p<0.01) due to the inhibition of Ras activity in RVLM. A Ras inhibitor decreased the activity of MAPK (‐21%, n=5, p<0.01), ERK (‐33%, n=5, p<0.01), caspase‐3 (‐42%, n=5, p<0.01) and expression of p53 in RVLM of SHRSP. ICV injection of caspase‐3 inhibitor for 14 days also significantly decreased BP, HR, SNA and activity of caspase‐3 in RVLM.

Conclusion

These results suggest that Ras‐activated caspase‐3 through MAPK and p53 in RVLM increases SNA in SHRSP, which might contribute to the impaired autonomic control in hypertension.
Elimination of nucleus of the solitary tract (NTS) neurokinin‐1 receptor (NK1R) expressing neurons enhances arterial pressure response to acute restraint, but attenuates enhancement of the arterial pressure response and adaption of post‐restraint hypotension during sub‐acute restraint in spontaneously hypertensive rats.
Authors: Bechtold Andrea G , Bonham Ann C
Published: June 2021

Cardiovascular hyperactivity to stress can predict subsequent cardiovascular disease in humans. Spontaneously hypertensive rats (SHR) also exhibit enhanced stress reactivity. Experiments were performed to test the hypothesis that the mean arterial pressure (MAP) response to restraint is dependent in part on nucleus of the solitary tract (NTS) neurokinin‐1 receptors (NK1R) in SHR. The NK1R targeted neurotoxin, stable substance‐P saporin (SSP) or saporin vehicle (SAP) was microinjected into the NTS to ablate NK1R neurons in SHR implanted with telemetry transmitters to measure MAP. Three weeks after NK1R neuron ablation, the MAP response to 1 hr of restraint was measured. SSP (n=10) significantly enhanced the MAP response during acute (day 1) restraint (p<0.05, area under the curve=2447 ± 158 for SSP vs 2167±42 mmHg/60 min for SAP; n=11). After restraint, MAP fell below pre‐stress values (post‐restraint hypotension; PRH) in both groups, with a shorter duration in the SSP rats (p<0.05). On subsequent days (sub‐acute restraint; 2‐4 d) the MAP response during restraint increased and PRH duration was reduced in SAP, but not SSP rats (p<0.05). We conclude that in SHR NTS NK1R neuron ablation enhances the MAP response during acute restraint, but during sub‐acute restraint attenuates the normal enhancement of the MAP response during restraint and adaption of PRH after restraint. (Support: HL‐67183)
Effects of local microejection of biogenic amines into the pre‐Botzinger complex (pBC) and adjacent ventral respiratory column (VRC) on the canine breathing pattern
Authors: Radocaj Tomislav , Mustapic Sanda , Stucke Astrid G. , Stuth Eckehard A. , Zuperku Edward J.
Published: June 2021

Previous studies suggest the presence of serotonin (5HT), dopamine (DA), and adrenergic receptors within the pBC region, but where these neuromodulators produce their principal effects on breathing remains unclear. The effects of local microinjections of 5HT (5mM), norepinephrine (NE; 5mM) and DA (5mM) into the VRC including the pBC on inspiratory (TI) and expiratory durations (TE) and peak phrenic activity (PPA) were studied in decerebrate, vagotomized, ventilated, paralyzed dogs (n=6). The locations of the VRC and pBC were determined by neuronal recordings and phrenic responses to glutamate agonist DLH (20mM, ~40 nl) microinjections. Unilateral injections (~100 nl) were made in 1 mm increments from the obex to 7mm rostral. The selective 5HT2a agonist (DOI, 0.2mM) and 5HT1a agonist (8OH‐DPAT, 0.2mM) were used in a similar protocol (n=7) and subsequently given iv. All amine agonists produced no significant changes in TI, TE, and PPA at any location within the VRC, while DLH injections produced significant changes. DOI (256 mcg/kg iv) produced a 10.3±5.4% increase in TI with no changes in TE and PPA, while 8OH‐DPAT (74 mcg/kg iv) decreased TI and TE to 67.1±4.5% and 62.3±7.2%, respectively with no change in PPA. These data suggest that the exogenous biogenic amines produce their effects on the breathing pattern at locations outside the VRC and pBC in dogs. Supported by VA Med. Res. and NIH GM059234 funds.
Moderate hyperoxia inhibits glomus cell proliferation in the carotid body of neonatal rats
Authors: Broge Thomas A , Dmitrieff Elizabeth F , Piro Samantha E , Bavis Ryan W
Published: June 2021

Rats exposed to hyperoxia during the perinatal period exhibit smaller carotid bodies with fewer O2‐sensitive glomus cells as adults (Erickson et al., 1998, J. Physiol. 509: 519‐26). To study the time course and mechanisms underlying these changes, rats were exposed to 60% O2 or room air from birth and their carotid bodies were harvested at various postnatal ages (P1‐P7, P14) for histological analysis (n=6 per age per treatment). The carotid bodies of hyperoxia‐treated rats were significantly smaller than those of age‐matched controls beginning at P4. Additional pups were injected with BrdU at P1, P3 and P5 and assayed for cumulative cell division 24 hours later (i.e., at P2, P4 and P6). The proportion of glomus cells that were BrdU‐positive was significantly reduced by hyperoxia at P4 (9±1% (SEM) vs. 28±3%; P<0.001); a similar trend was observed at P2 but not at P6. TUNEL staining was used to assess cell death at P2, P4 and P6. The proportion of glomus cells that were TUNEL‐positive was low at all ages but somewhat greater in hyperoxia‐treated rats (0.9±0.1% vs. 0.3±0.1% across ages; P<0.01). These data indicate that hyperoxia attenuates postnatal growth of the carotid body by inhibiting glomus cell proliferation during the first few days of exposure, although hyperoxia‐induced apoptosis may also contribute to the smaller carotid body size. Supported by NIH grant P20 RR‐016463 (Maine INBRE).
Influence of long‐term facilitation on timing, amplitude, and inspiratory neural network complexity in urethane‐anesthetized P20‐21 neonatal Sprague Dawley rats
Authors: Reid Inefta M , Solomon Irene C
Published: June 2021

Long‐term facilitation (LTF) is a form of respiratory plasticity, which is expressed as a persistent increase in inspiratory motor output following intermittent, but not continuous, hypoxia. We have recently begun to examine LTF‐related changes in the timing, magnitude, and inspiratory neural network complexity (quantified by approximate entropy ApEn analysis) in neonatal (ages P12‐P18) rats. Our previous work has revealed that respiratory LTF is characterized by an enhancement of burst frequency due to reductions in both TI and TE (except in P12 rats), an increase or no change in burst amplitude, and an age‐dependent modulation in ApEn, such that ApEn values were reduced in P12‐P13 rats and increased in P14‐ P18 rats; the peak responses occurred at = 50 min in P12‐P13 rats and at = 45 min in P14‐ P18 rats. The LTF‐related changes in older neonatal rats remain to be identified. Therefore, we examined these features during LTF in 7 spontaneously breathing urethane‐anesthetized P20‐21 rats. In this case, respiratory LTF was characterized by an increase in burst frequency, an ~10‐20% decrease in burst amplitude, and variable effects on ApEn, such that ApEn values were decreased in P20 rats and increased in P21 rats. Regardless, the peak changes in ApEn values were noted at ~40 min for both age groups. Our findings suggest that respiratory LTF‐related changes are developmentally regulated. Supported by NS045321 and NS049310
Neonatal induced mild diabetes: influence on rat development and behavioral activity
Authors: Kiss Ana Carolina Inhasz , Sinzato Yuri Karen , Spada Ana Paula Machado , Bueno Aline , Iessi Isabella , Sakamoto Silvia , Saito Felipe , Kempinas Wilma De Grava , Rudge Marilza Vieira Cunha , Damasceno Débora Cristina
Published: June 2021

The aim was at evaluating the effect of neonatal mild diabetes induced by streptozotocin on development and behavioral activity of female Wistar rats. At birth, 81 female rats were randomly assigned into two groups: Control (n=20) received citrate buffer (subcutaneous route ‐ sc.) and STZ (n=61) received streptozotocin (STZ ‐ 100 mg/kg ‐ sc.). Glycemia was measured at postnatal days (PND) 35, 84 and 120. Physical development was daily evaluated. At PND 10 and 21, body weight (g), length (cm), and anogenital distance (mm) were measured and anogenital index was obtained. Behavioral activity was evaluated in an open field at PND 10, 21 and 75. Streptozotocin administration in the neonatal period of Wistar rats caused mild hyperglycemia in adulthood with no effects on physical development, although resulting in reduced body length and anogenital distance. Rats with mild hyperglycemia intensity (STZ group) also presented reduced locomotor and exploratory behavior, which are similar to the results presented in behavioral studies of severe diabetic rats. These data showed that even a modest hyperglycemia is enough to impair behavioral activity. Financial support by Capes.