bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic and ventilatory changes upon exposure to hypoxia and hypercapnia in neonatal fat‐tailed dunnarts (Sminthopsis crassicaudata)
Authors: Simspon Shannon J , Frappell Peter B
Published: June 2021

The fat‐tailed dunnart is born small and developmentally immature (mass 13 mg, gestation 13.5 days). At birth, the lungs contain only a few air sacs, and the skin is the only organ of gas exchange until the onset of continuous ventilation and a functional residual capacity (FRC) is established. We examined ventilation and metabolism during normoxia/normocapnia in neonatal dunnarts ranging from postnatal day (P) 0 to P23. Rhythmic ventilation, with the characteristic post‐inspiratory pause, was not detected until several days after birth. We also investigated the effects of hypercapnia (5%CO2) and hypoxia (10% O2) on ventilation and metabolism. The newborn dunnart responded to acute hypoxia by hyperventilation, which was achieved mostly by a decrease in metabolic rate (hypometabolism). This hypometabolism led to marked decrease in minute ventilation (VE) after several minutes of exposure and resulted in prolonged periods of apnoea. As the pouch young age, both metabolic and ventilatory components contribute to the hypoxic hyperventilation. Upon exposure to hypercapnia, the non‐breathing P0 dunnarts appear to have a similar hypometabolic response. The expected response of hyperventilation via an increase in VE, predominantly associated with an increase in tidal volume, was observed in the P5 and older neonates. Exposure to hypercapnia also led to an increased stability in the breathing pattern and frequency of augmented breaths or sighs.
Effects of acute heat stress on skeletal muscle gene expression associated with energy metabolism in rats
Authors: Sanders Sara R , Cole Lori C , Flann Kyle L , Baumgard Lance H , Rhoads Robert P
Published: June 2021

Study objectives were to determine the effects of acute heat stress (HS) on the expression of genes associated with energy metabolism in type I and II skeletal muscles. Male Sprague Dawley rats (n = 12; 9 mo) were randomly assigned to either thermal neutral (TN; 21.1°C) or HS conditions (39.4°C) for 6 h. Rats were anesthetized and the soleus (type I; oxidative) and tibialis anterior (TA; type II; glycolytic) muscles were collected. Total RNA was isolated and cDNA generated for real‐time PCR analysis (iQ5 Real‐Time PCR detection system). Exposure to HS tended to increase soleus and decrease TA AMP‐activated protein kinase (AMPK) mRNA abundance. Hyperthermia increased and tended to increase PPAR‐γ Co‐activator ‐1α (PGC‐1α) gene expression in soleus and TA, respectively. Pyruvate dehydrogenase kinase 4 (PDK4) mRNA abundance was increased by HS in the soleus but was unaltered in the TA. Lactate dehydrogenase‐α (LDHα) gene expression tended to increase in TA during HS but LDHα mRNA abundance did not differ in soleus between treatments. In conclusion, exposure to HS induced the differential expression of several energy metabolism genes based on muscle type (soleus versus TA). Moreover, changes in gene expression are consistent with impaired cellular energy status, perhaps due to mitochondrial dysfunction, in oxidative compared to glycolytic muscle fibers during hyperthermia.
Fatigue preconditioning increases fatigue resistance in mouse FDB using a different intracellular signalling pathway to ischemic preconditioning.
Authors: Boudreault Louise , Cifelli Carlo , Bourassa Francois , Renaud Jean‐Marc
Published: June 2021

The objective of this study was to determine the nature of the intracellular signalling pathway that regulates fatigue preconditioning (FPC) whereby one fatigue bout (FAT1) acutely increases the fatigue resistance of muscle during a second fatigue bout (FAT2). All fatigue bouts were elicited with one tetanic contraction every s for 3 min and FAT2 was elicited 60 min after FAT1. The decreases in peak tetanic Ca2+i and force were significantly slower while the increases in unstimulated Ca2+i and force were significantly less during FAT2 than during FAT1. The differences between FAT2 and FAT1 were even greater when KATP channels were blocked with 10 µM glibenclamide. Ischemic preconditioning (IPC) is a phenomenon in which short, non‐ damaging ischemic periods increase muscle resistance to the damaging effects of a long and damaging ischemic period. While adenosine, sarcolemmal and mitochondrial KATP channels, protein kinase C and reactive oxygen species have all been implicated in IPC, the addition of their specific blockers during FAT1 did not prevent the increased fatigue resistance during FAT2. We therefore conclude that IPC and FPC have different intracellular signalling pathways.
Slowed convective blood flow delivery speeds the onset of fatigue in highly‐oxidative skeletal muscle
Authors: Hernandez Andres , Goodwin Matthew L. , Gladden L. Bruce
Published: June 2021

It was the purpose of this study to examine the role of slowed blood flow kinetics at the onset of contractions on fatigue characteristics in highly‐oxidative skeletal muscle. A step change in metabolic rate was elicited by stimulating canine gastrocnemius‐plantaris muscles (n=6) via their sciatic nerves (6‐8 V, 0.2 ms duration, 50 Hz, 200 ms train) at a rate of 1/2 s. With arterial O2 concentration maintained constant, in random order trials, blood flow tau was set at a control transition speed between rest and contractions (determined in pilot work) of 20 s (CT20) or was slowed to tau values of 45 s (EX45) and 70 s (EX70). Resting and steady‐state (4 min of contractions) blood flows were not altered between the transition conditions. Peak force was not significantly different (p>0.05) between conditions (mean±SD; CT20=293±64 N; EX45=265±64 N; EX70=256±56 N). Final normalized force was also not significantly different (p>0.05) between conditions (CT20=87±6; EX45=88±7; EX70=88±11 %). Time from peak force to 50% of final force was significantly faster (p=0.0163) for EX45 (76±25 s) in comparison to CT20 (103±12 s). Time to 50% of final force was also significantly faster (p=0.0195) for EX70 (72±19.391 s) vs. CT20 while EX45 and EX70 were not significantly different. To our knowledge, these are the first experiments to alter only the time course of blood flow at contractions onset without changing the baseline and steady‐state flows, and our data suggest that fatigue occurs more rapidly with slowed blood flow only in the transition period at the onset of contractions in highly‐oxidative skeletal muscle.
Fatty acids increase toll‐like receptor‐4 gene expression, protein content in a saturation‐dependent manner in C2C12 skeletal muscle cells
Authors: McMillan Ryan , Frisard Madlyn , Hulver Matthew
Published: June 2021

Growing evidence suggests that obesity, and associated metabolic dysregulation, occurs in concert with chronic low‐grade inflammation. Our laboratory has reported that toll‐like receptor‐4 (TLR4) expression, an inflammatory mediator, is elevated in the skeletal muscle of obese humans and associated with reduced fatty acid oxidation and increased lipid synthesis. The purpose of this study was to examine the role of saturated vs. monounsaturated fatty acids on TLR4 transcription and function. Our hypothesis was that saturated fatty acids would sensitize the TLR4 pathway via increased receptor content. To this end, skeletal muscle cells were incubated in the presence of varying ratios of palmitate (16:0) and oleate (18:1) to examine the effects of fatty acid saturation on TLR4. Fatty acid exposure resulted in a significant and stepwise increase in TLR4 mRNA and protein content with increasing palmitate concentration. To determine if the fatty acid effect on TLR4 was translated into a functional consequence, we measured the effect of TLR4 activation on substrate metabolism. As the degree of fatty acid saturation was increased in the media, the effect of TLR4 activation on glucose and fat oxidation was exacerbated. In conclusion, exposure to a lipotoxic environment sensitizes the TLR4 pathway in skeletal muscle and suggests that this is accomplished via a mechanism that is fatty acid saturation specific.
Alterations in skeletal muscle contractile force following acute exposure to thromboxane A2.
Authors: Touchberry Chad D. , Stone Jessica K. , Brotto Leticia S. , Brotto Marco P. , Wacker Michael J.
Published: June 2021

Thromboxane A2 (TxA2) has traditionally been associated with platelet aggregation and vasoconstriction of the microcirculation. A variety of inflammatory states such as aging and obesity are associated with increased circulating levels of TxA2. Our laboratory has recently shown that TxA2 can cause an increase in the magnitude and frequency of intracellular calcium transients in cardiac myocytes and significantly alters ventricular force generation. Because of the physiological cross‐talk between cardiac and skeletal muscles, we sought to elucidate the effects of TxA2 on skeletal muscle contractility. Ex‐vivo, slow‐twitch soleus (Sol) muscles from adult mice were contracted using both twitch (1 Hz) and titanic (40 Hz) stimulations. Following equilibration, Sol muscles were incubated in the presence of 10 µM U46619 (a known TxA2 mimetic). Muscle twitches showed a significant rise in baseline and a corresponding decline in peak twitch height following U46619 administration; however, muscles tested under tetanic stimulation showed an increase in force production following U46619. These data suggest TxA2 is an important modulator of skeletal muscle force production and may play a role in altering calcium handling in skeletal muscle. These findings may lead to a better understanding of altered muscle function during inflammatory disease states and fatigue‐related myopathies.
Disruption of a conserved tryptophan at the base of TM1 in αENaC confers voltage‐sensitivity to the channel
Authors: Pochynyuk Oleh , Mironova Elena , Boiko Nina , Kucher Volodymyr , Staruschenko Alexander , Stockand James David
Published: June 2021

ENaC activity is critical for Na+ reabsorption across many epithelial barriers. ENaC is a highly Na+‐selective, non‐voltage gated in the ENaC/Deg superfamily. Here, we identify a conserved tryptophan residue at the base of TM1 that dramatically affects ENaC gating properties. Targeted substitution of W112 to non‐polar residues (F, I, C, G) in mouse αENaC decreased ENaC activity by 3 fold, whereas substitution to polar residues (K, E) resulted in non‐functional channels. Decreases in activity were associated with changes in ENaC Po by reduction of mean open time. Surprisingly, ENaC Po became voltage‐sensitive with mutant channel having increased Po upon hyperpolarizing voltages. In addition, macroscopic ENaC currents had fast (approximately 200 msec) activation and deactivation time constants at negative and positive voltages. These time constants were weakly voltage‐dependent. Fitting G‐V curves with the Boltzmann equation established V½ at approximately ‐150 mV. Decreasing Na+ concentration to 2 mM did not affect voltage‐sensitivity, excluding Na+ self‐inhibition as a mechanism. Ca2+, Mg2+ and Cl‐ also had no pronounced effect on gating properties or voltage‐sensitivity of the mutant channel. We proposed that this critical W provides stability to TM1 and, thus, the channel pore. Disrupting this causes a change in ENaC gating properties and voltage‐sensitivity.
Parameter Estimation for Models of NH4+ Transport by the Renal Na‐K‐2Cl Cotransporter
Authors: Marcano Mariano , Yang Hun‐Mo , Nieves‐González Aniel , Clausen Chris , Moore Leon C.
Published: June 2021

An optimization algorithm was used to compute parameter values for kinetic models of NH4+ transport by the renal Na‐K‐2Cl cotransporter (NKCC2). The optimization method finds model off‐binding and translocation rate constants by minimizing the difference between model unidirectional fluxes and published data on NH4+‐ dependent 86Rb+ uptake by the NKCC2 cotransporter in transfected Xenopus oocytes. By using different symmetry assumptions, which reduces the number of parameters, five different models were optimized. The computed parameters for each model yielded fluxes consistent with the experimental curves, and the fittings of the five models exhibited residual errors that were not significantly different from each other. However, a systematic exploration of the parameter space revealed that, in some models, different parameter sets yield essentially identical fits to the data. With the identified parameters, the NKCC2 models show K+/NH4+ exchange, and enhancement of Na+ uptake by NH4+ binding when external [K+] is low. Further, for the A, B, and F isoforms, the optimization method identified parameter sets that differ only in off‐binding affinities, a result that is consistent with a published mutagenesis analysis of the molecular and structural bases for differences in 86Rb+ uptake between isoforms.

This research was supported in part by NIH grants SC1GM084744 and DK042091.
Candesartan restored the altered renal insulin receptor protein and cytokine profile, and reduces nephropathy in the obese Zucker rat despite its propensity to worsen diabetes
Authors: Tiwari Swasti , Rash Arjun , Ecelbarger Carolyn
Published: June 2021

Candesartan (CAN), an angiotensin type 1 receptor (AT1R) antagonist, exhibit renoprotective effect in obese Zucker rats (OZ). We have reported downregulated renal insulin receptor (IR) in hyperinsulinemic OZ. IR could attenuate inflammation via NO production. Cytokines are central to the pathology of nephropathy. To determine the effect of chronic CAN therapy on cytokine expression and their correlation with IR in OZ kidney, male lean (LZ) and obese (OZ) Zucker rats were treated without or with CAN (25 mg CAN/kg·diet) (n= 6/treatment/body type) for 12 weeks. Histochemical staining revealed attenuated nephropathy by CAN in OZ. CAN significantly reduced urinary albumin and plasma creatinine levels in OZ. Surprisingly, glucose tolerance was worsened in CAN‐treated OZ. ELISA‐based cytokine array in the whole kidney revealed that OZ had a marked increase expression of monocyte chemoattractant protein‐1 (MCP‐1, 202 ± 58 vs. 68 ± 6 pg/ml) and interleukin (IL‐1 β, 235 ± 30 vs. 152 ± 11 pg/ml) relative to LZ. CAN treatment restored these levels to LZ levels. Furthermore, IL‐10, a cytokine important for inhibiting the synthesis of pro‐inflammatory cytokines, was significantly lower in OZ (135 ± 9 vs. 250 ± 9 pg/ml) and CAN treatment restored its level to LZ. Surprisingly, other proinflammatory cytokines including IL‐1α and IL‐6 were significantly below LZ level in OZ and were restored to lean levels by CAN. In addition, renal IR protein levels positively correlated with IL‐10 and negatively with IL‐1 β (p=0.02). These data demonstrate that blockade of AT1R reduces MCP‐1 and IL‐1 β and upregulate IL‐10. The correlation of IR with inflammatory responses suggests either inflammation reduced IR or IR action is important in preventing inflammation. Additional studies are required. NKF (ST) and NIH (CE)
Changes in Renal Interstitial Fluid Pressure Do Not Mediate Arterial Pressure‐induced Medullary NO Production
Authors: Jin Chunhua , Cowley Allen W.
Published: June 2021

The present study examined whether renal outer medullary NO production, as reflected by changes in NO3+NO2, would be enhanced by increased renal perfusion pressure (RPP) and whether this is related to associated changes of renal interstitial fluid pressure (RIFP). Two groups of male Sprague Dawley rats (10 wks) were compared; intact renal capsule (IN; n=7) and decapsulated (DC; n=6). The left kidney was denervated, a linear microdialysis probe implanted into the outer medulla, and urine collected from a catheter in the left ureter. Medullary interstitial and urinary excretion of NO3+NO2 were determined. An adjustable aortic occluder proximal and distal to the left renal artery was used to increase RPP from 80 to 110 to 140 mmHg. As RPP increased, medullary NO3+NO2 increased significantly in both groups (IN= 9.2+2.8, 13.8+3.9 to 16.1+3.9; DC= 8.9+2.4, 15.7+3.2 to 16.9+3.3 uM) as did urinary NO3+NO2 excretion (IN= 1.6+0.9, 7.0+2.9 to 11.1+4.8; DC= 1.1+0.3, 6.6+0.9 to 7.8+1.3 nmol/min/g kwt). Only natriuresis and diuresis were significantly blunted in the DC group. We conclude that acute increases in RPP within the autoregulatory range induces a concomitant increase in intrarenal NO production independent of the RIFP‐determined pressure‐natriuresis component and may be a consequence of increases of medullary blood flow.
The role of central AT1 receptors in age related changes of hypothalamic redox signaling and adrenomedullary tyrosine hydroxylase expression
Authors: Basgut Bilgen , Woods Mary E , Cudykier Idan , Whidden Melissa A , Rodriguez Michael , Erdos Benedek , Tumer Nihal
Published: June 2021

The aim of this study was to test the role of angiotensin II (AngII)‐mediated mechanisms in age‐related changes of hypothalamic catecholamine biosynthesis, redox signaling and sympathetic nervous system (SNS) activation. Young (5 mo, n=6) and old (27 mo, n=6) F‐344xBN rats were infused intacerebroventricularly with either artificial CSF or losartan (15 µg/hour) for 3 days using osmotic minipumps. Level of hypothalamic tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine biosynthesis, decreased significantly with age, and in response to losartan both in young and old animals. In addition, MnSOD, catalase and NADPH oxidase‐p47 levels were decreased in the hypothalamus with age, and also with losartan treatment in both age groups. Adrenomedullary TH, an indicator of SNS activity, increased significantly with age, but decreased after losartan infusion in both young and old rats reversing the age‐related TH upregulation. These results suggest that central AT1 receptor signaling play an important role in the regulation of hypothalamic redox signaling and SNS regulation.
Central administration of angiotensin‐(1‐7) markedly reduces the tachycardia evoked by acute psychological stress exposure
Authors: Lima Augusto Martins , Xavier Carlos Henrique , Santos Robson Augusto Souza , Fontes Marco Antônio Peliky
Published: June 2021

There is now extensive evidence that the activity of brain RAS is important in the control of anxiety‐related behaviors. In this study we investigated possible central effects of the heptapeptide angiotensin‐(1‐7) [Ang‐(1‐7)] in the cardiovascular reactivity to acute psychological stress. Under tribromoethanol anesthesia (250 mg/Kg i.p.) male wistar rats (?300g) received guide cannula into cerebral ventricle (i.c.v.) for microinjections of drugs. Four days later, a catheter was placed into femoral artery for recording cardiovascular parameters. After 24 h, three different doses of Ang‐(1‐7) [50, 100 and 200 pmol/2 ?l (n=5 each dose)] or vehicle [control, NaCl 0.9%, 2 ?l (n=6)] was administered i.c.v. and rats were submitted to air jet stress (10 L/min ‐ 10 min). In control experiments, the tachycardia evoked by air stress (? 148 ± 18 bpm) was markedly attenuated after 100 and 200 pmol i.c.v. injections of Ang‐(1‐7) (? 80 ± 18 bpm and ? 75 ± 29 bpm, p<0.05, respectively). This attenuation was observed throughout the 10 min stress trial. After treatment with these doses of Ang‐(1‐7), heart rate during the post‐stress recovery period was also lower than that seen after treatment with vehicle (vehicle 449 ± 25 bpm; Ang‐(1‐7) 100 pmol 382 ± 11 bpm; 200 pmol 377 ± 22 bpm; p<0.05) . Changes in blood pressure caused by air jet stress (21 ± 11 mmHg after vehicle) were more variable and not significantly altered by any dose of Ang‐(1‐7). We conclude that Ang‐(1‐7) might act in central regions attenuating the cardiac response evoked by acute psychological stress. Financial Support: CNPq, FAPEMIG, PRONEX.
Cardiovascular responses to voluntary and forced underwater submergence in trained and untrained rats
Authors: McCulloch Paul F. , DiNovo Karyn M.
Published: June 2021

The mammalian diving response consists of apnea, a parasympathetically‐induced bradycardia and a sympathetically‐induced increase in peripheral resistance. Although initiation of this autonomic response is a trigeminally mediated reflex, we wanted to determine if the magnitudes of the cardiovascular responses to diving are altered by the nature of the dive or previous diving experience. Six rats were trained in both voluntary and forced diving, while 6 other rats received no training. All rats were implanted with telemetric transmitters to record mean arterial blood pressure (BP, in mm Hg) and heart rate (HR, in beats per minute). In trained rats undergoing voluntary dives HR immediately decreased from 468 ± 14 to 128 ± 16 and BP increased from 145 ± 1 to a maximum of 169 ± 4, while in untrained rats HR immediately decreased from 468 ± 21 to 98 ± 11 and BP increased from 136 ± 4 to a maximum of 173 ± 4. In trained rats undergoing forced dives HR immediately decreased from 442 ± 4 to 125 ± 16 and BP increased from 132 ± 4 to a maximum of 191 ± 8, while in untrained rats HR immediately decreased from 452 ± 14 to 115 ± 9 and BP increased from 125 ± 4 to a maximum of 194 ± 5. We conclude that the type of dive and previous experience alters the magnitude of the HR and BP responses to underwater submergence. This suggests that cortical input can modify both the parasympathetic and sympathetic limb of this autonomic reflex. Supported by NIH R15 HL080007.
Glutamate regulates local and global Ca2+ signals in myocytes of piglet brain slice arterioles through astrocyte‐ and heme oxygenase‐dependent mechanisms
Authors: Zhao Guiling , Xi Qi , Umstot Edward , Leffler Charles W , Jaggar Jonathan H
Published: June 2021

Glutamate is the principal cerebral excitatory neurotransmitter and dilates cerebral arterioles to match blood flow to neural activity. Arterial contractility is regulated by local and global Ca2+ signals that occur in myocytes, but modulation of these signals by glutamate is poorly understood. Using high‐speed confocal imaging we measured glutamate regulation of Ca2+ signals that occur in myocytes within arterioles of piglet tangential brain slices. Glutamate elevated Ca2+ spark frequency ~188% and reduced global intracellular Ca2+ concentration ([Ca2+]i) to ~76% of control, but did not alter Ca2+ waves. Cerebral arteriole isolation from brain slices abolished glutamate‐induced Ca2+ signal modulation. In slices treated with L‐AAA, a glial toxin, glutamate did not alter Ca2+ sparks or global [Ca2+]i, but activated Ca2+ waves. This shift in Ca2+ signal modulation by glutamate did not occur in slices treated with D‐AAA, an inactive isomer of L‐AAA. In the presence of CrMP, a heme oxygenase blocker, glutamate inhibited Ca2+ sparks and Ca2+ waves and did not alter global [Ca2+]i. Collectively, these data indicate that glutamate can modulate local and global Ca2+ signals in arteriole myocytes via mechanisms that require astrocytes and heme‐oxygenase. In the absence of astrocytes or functional heme oxygenase, effects of glutamate on arterial Ca2+ signals were absent or the opposite of those in the intact system.
Cerebral cortex functional hyperemia and plasticity in the rat during a skilled reach task
Authors: Hart Lucas M. , Harder David
Published: June 2021

Neurons of the caudal forelimb area (CFA) of the rat cerebral cortex are known to undergo synaptogenesis and motor map reorganization as a result of skilled reach training. This is used as a motor learning paradigm. Cerebral neurons signal through astrocytes to initiate an increase in cerebral blood flow during local brain activation, which is termed functional hyperemia (FH). We investigated whether FH magnitude, or the astrocyte marker glial fibrillary acidic protein (GFAP), show changes in the CFA related to skilled reach training. Adult rats were trained to reach for food pellets using their preferred limb for 10 days. Blood flow was measured over the contralateral CFA on days 1 and 10 using laser Doppler flowmetry. On the eleventh day the brain was excised and the CFA dissected bilaterally for Western blot analysis of GFAP levels. Our data suggest that there may be a positive correlation between FH magnitude and motor learning. Moreover, preliminary data suggest that GFAP levels may be higher in the cortex contralateral to the preferred limb in rats that demonstrated motor learning when compared to those that did not exhibit motor learning. Overall, we observed that cerebral blood flow and astrocyte structure or density may undergo plasticity in the CFA under motor learning conditions. This hypothesis adds to the complexity of how the functional units of the brain adapt when acquiring motor skills. Funded by National Institutes of Health grant P01 HL59996
Role of ERK1/2 and Rho A/Rho kinase in PKC‐induced contractions in ovine cerebral arteries: A developmental study
Authors: Mittal Ashwani , Goyal Ravi , Shi Lijun , Wedhas Nia , Longo Lawrence D.
Published: June 2021

Objective

We tested the hypotheses that developmental maturation is associated with significant alterations in PKC mechanisms in Ca2+‐independent vascular contraction. In fetal and adult ovine cerebral arteries, in response to PKC stimulation, we quantified both total and phosphorylated levels of several proteins and tension and intracellular [Ca2+]i.

Results

In both adult and fetus, PDBu‐induced contractions were 65% and 25% of Kmax, respectively, and ERK1/2 inhibition by U‐0126 decreased these contractions 40%. The ROCK inhibitor Y‐27632, decreased the response 50% in fetus but not adult. In adult cerebral arteries, PDBu increased phosphorylation of ERK1/2, ERK1/2‐dependent CaDSer789, CPI‐17Thr38, but not Rho A. In contrast, in fetal cerebral arteries activated Rho A, but not CaDSer789 or CPI‐17Thr38. By co‐immunoprecipatation, in adult, but not fetal, cerebral arteries PDBu‐simulated interaction between PKCα andCPI‐17, and PKCα and caldesmon.

Conclusions

In cerebral arteries, maturation with increasing myofilament Ca2+ sensitivity by MLCP regulation is associated with a transition from a PKC‐ERK1/2, Rho A‐dependent pathway in the fetus to one of CaDSer789‐ and CPI‐17Thr38‐dependent phosphorylation and increased myofilament Ca2+ sensitivity in the adult. In the immature organism, these differing signaling pathways may be associated with dysregulation of cerebral blood flow. (USPHS HD 03708)
Neuroprotective effect of adenoviral catalase gene transfer in cortical neuronal cultures
Authors: Gáspár Tamás , Domoki Ferenc , Lenti Laura , Institoris Ádám , Snipes James A , Bari Ferenc , Busija David W
Published: June 2021

Reduced availability of reactive oxygen species is a key component of neuroprotection against toxic stimuli. Recently, we have shown that the hydrogen peroxide scavenger catalase plays a central role in delayed preconditioning induced by the mitochondrial ATP‐sensitive potassium channel opener BMS‐191095 (Gáspár et al. JCBFM 28 (6): 1090‐1103, 2008). The aim of our present experiments was to investigate the neuroprotective effect of catalase in vitro using an adenoviral catalase gene transfer protocol. To induce catalase overexpression, cultured rat cortical neurons were incubated with the adenoviral vector Ad5CMVcatalase and control cells with Ad5CMVntLacZ for 24 h. Gene transfer increased catalase protein levels and activity, but did not influence other antioxidants. Catalase transfection did not protect against glutamate excitotoxicity and oxygen‐glucose deprivation, but conferred a dose‐dependent neuroprotection against 6 h exposure to 80 μM hydrogen peroxide (viability: control, 20.70±1.96%; LacZ 10 pfu/cell, 15.00± 1.66%; catalase 1 pfu/cell, 57.60±5.46%*; catalase 2 pfu/cell, 104.90±1.74%*; catalase 10 pfu/cell, 111.20±1.92%*; *p<0.05 vs. control; mean±SEM). Finally, the protection could be antagonized using the catalase inhibitor 3‐aminotriazole. Our results support the view that enhancing cellular antioxidant capacity may play a crucial role in neuroprotective strategies.
Nitric oxide contributes to the augmented vasodilation during hypoxic exercise
Authors: Casey Darren P , Madery Brandon D , Elvebak Rachel L , Curry Timothy B , Eisenach John H , Wilkins Brad W , Joyner Michael J
Published: June 2021

We previously reported that augmented skeletal muscle vasodilation during mild hypoxic forearm exercise includes beta‐adrenergic mechanisms. Furthermore, hypoxic vasodilation at rest is known to include beta‐adrenergic mediated nitric oxide (NO) production. Therefore, we tested the hypothesis that NO contributes to augmented vasodilation with hypoxic exercise. Twelve subjects (6M/6F; 26 ± 2 years) breathed hypoxic gas to titrate arterial O2 saturation to 80% while remaining normocapnic via a rebreath system. Subjects performed incremental forearm exercise (10% and 20% of maximum) during saline infusion (control) and NO synthase inhibition (NG‐monomethyl‐L‐arginine; L‐NMMA) under normoxic and hypoxic conditions. Forearm vascular conductance (FVC; ml/min/100mmHg) was calculated from blood flow (ml/min) and blood pressure (mmHg). During the control condition, the rise in FVC (Δfrom normoxic baseline) due to hypoxic exercise was 211 ± 23 and 367 ± 34 (10% and 20% respectively). Infusion of L‐NMMA during hypoxic exercise attenuated ΔFVC at 10% (174 ± 20; P < 0.001) and 20% (317 ± 32; P < 0.01) compared to control condition. Our data indicate that NO contributes to the augmented vasodilation observed during hypoxic exercise.

Supported by NIH HL‐46493 (MJJ) and AR‐55819 (DPC) and by CTSA RR‐024150.
The Effect of Reactive Oxygen Species on Bone Mineral Density and Bending Stiffness in Superoxide Dismutase (SOD) Knockout Mice
Authors: Smietana Michael J , Kaushik Amit , Arruda Ellen M , Faulkner John A , Larkin Lisa M
Published: June 2021

CuZn‐SOD knockout mice Sod1−/− have elevated oxidative stress and decreased body mass, musculoskeletal mass , and whole muscle strength compared to wild‐type mice Sod1+/+. We tested the hypothesis that bone bending stiffness (EI; Nmm2) is also diminished in the Sod1−/− compared to the Sod1+/+ mice. Femurs were obtained from male and female Sod1+/+ and Sod1−/− mice at 8 mo of age (n = 3 per gender and genotype) and analyzed for bone mineral density (BMD; mg/cc) using Micro Computed Tomography and bending stiffness using a three‐point bending test. There were no significant differences in BMD or EI with respect to gender in either genotype. Sod1−/− mice exhibited a significant decreases in BMD and EI in both males and females as compared with Sod1+/+mice, P=0.004 and 0.005 respectively.

Values are Means + SD. These data suggest that like muscle strength, the increase in reactive oxygen species may play a role in decreasing bone bending stiffness and mineralization in the Sod1−/− mice. Supported by PO1 AG20591.
Intracellular localization of TDAG51 modulates its effect on cell death
Authors: Al‐Bayati Hiam Kamil , Dickhout Jeffrey G. , Traunt Ray , Austin Richard C.
Published: June 2021

Our previously findings indicate that T‐cell death associated gene 51 (TDAG51) contributes to cell death in atherosclerotic lesions. To determine if these cell death effects are mediated by TDAG51 intracellular localization, we performed sequence analysis of the TDAG51 cDNA to identify novel cellular targeting domains. This analysis revealed two putative motifs; a nuclear exiting signal (NES) (70‐vasleppvkl‐80) and nuclear localization signal (NLS) (16‐krsdgllqlwkkk‐28). We hypothesized that these TDAG51 motifs play a critical role in its intracellular transport, thereby modulating its pro‐apoptotic effects. To investigate this phenomenon, VKL was mutated to AKA in NES to impair exiting of the TDAG51 protein from the nucleus to the cytoplasm and WKKK was mutated to WKAA in NLS to impair entrance of TDAG51 into the nucleus. Following transient transfection of the fused GFP constructs into HeLa cells, the cellular localization of these mutant TDAG51 proteins was assessed using confocal microscopy on fixed cells or visualized in living cells over a 2 to 48 hour time period. Our findings show that both motifs are involved in the intracellular transport of TDAG51. By determining cell viability, we were able to determine the predominant effects of nuclear TDAG51 on cell death. These studies provide a better understanding of the cellular motifs in TDAG51 that contribute to cell death. Supported by the CIHR (MOP‐74477).
Interleukin‐1 beta (IL‐1β) via inhalation or right atrial injection increases pulmonary substance P (SP) and capillary permeability with little effect on ventilation and airway resistance in anesthetized rats
Authors: Zhuang Jianguo , Xu Junyang , Zhang Cancan , Xu Fadi
Published: June 2021

Bronchopulmonary sensory C‐fibers (PCFs), when activated, produce bronchoconstriction and apnea, and increase pulmonary SP and capillary permeability (CP). Injection and inhalation of IL‐1β, a pro‐inflammatory cytokine, into the airway and lung activate PCFs and elevate pulmonary CP, respectively. We therefore tested whether pulmonary IL‐1β affected ventilation (VE), airway resistance (RL), pulmonary SP, and CP. VE and RL were separately recorded in two groups of anesthetized rats before and after IL‐1β administration. Four hours later, bronchoalveolar lavage fluid (BALF) and lung tissue were collected to detect SP by EIA or CP by Evans‐blue extravasation. Our results showed that the right atrial injection (0.1 or 1.0 μg) and aerosol inhalation (0.5 or 5.0 μg/mL; 20‐60 s) of IL‐1β failed to change VE and RL, but markedly increased SP content in both BALF and lung tissue as well as the pulmonary CP (102%, 56%, and 110%; P < 0.01). We also tested whether the IL‐1β treatment would sensitize PCFs to enhance their apneic reflex, and found no difference in the apneic responses to right atrial bolus injection of capsaicin (4 μg/kg) between the rats pretreated with vehicle and IL‐1β as described above. These findings suggest that exogenous IL‐1β applied in the airway and lung does not affect VE and RL, but enhances pulmonary SP release and CP, likely via activation of PCFs in anesthetized rats. (Supported by NIH 74183)
Mast cell‐cholinergic nerve interaction in mouse airway
Authors: Weigand Letitia , Meeker Sonya , Myers Allen C , Undem Bradley J
Published: June 2021

Trachea were isolated from mice (C57BL/6J) that had been actively sensitized to ovalbumin (OVA) via 3 i.p. injections. OVA (10µg/mL) caused histamine release (≈50% total tissue content), and smooth muscle contraction that was rapid in onset and short‐lived (t½=<1 minute), reaching 26.5±2.2% of the maximum response to methacholine (n=18). OVA contraction was mimicked by 5‐hydroxytryptamine (5‐HT), and responses to both OVA and 5‐HT were sensitive to 10 µM‐ketanserin (5‐HT2receptor antagonist, n=5), and was strongly inhibited by atropine (1 µM, n=4). Epithelial denudation had little effect on OVA‐induced contraction (n=7). OVA failed to elicit histamine release or contractile responses in trachea isolated from sensitized mast cell‐deficient (sash ‐/‐) mice (n=3). Intracellular recordings of cholinergic neurons in mouse tracheal ganglia revealed a ketanserin‐sensitive 5‐HT‐induced depolarization (6.8 mV, n=6), and similar depolarization in response to OVA challenge. These data support the hypothesis that antigen‐induced contraction of mouse trachea is epithelium‐independent, and requires mast cell‐derived 5‐HT to activate 5‐HT2 receptors on parasympathetic cholinergic neurons. This leads to acetylcholine release from nerve terminals and airway smooth muscle contraction. This work was supported by the NIH.
Estrogen modulation of intracellular Ca2+ in airway smooth muscle inflammation
Authors: Townsend Elizabeth , Prakash Y S
Published: June 2021

Gender disparity in incidence and severity of asthma is clinically recognized. While estrogen‐induced vasodilation is well‐known, effects on airway smooth muscle (ASM) are controversial. In human ASM, we found that both estrogen receptors (ERs), ERα‐66 and ERβ‐47 are expressed, as are two truncated ERα isoforms (ERα‐36 and ERα‐47). In fura‐2 loaded human ASM cells, acute exposure to 17β‐estradiol (17βE2; 1nM) blunts intracellular calcium ([Ca2+]i) response to ACh and histamine, effects prevented by ER antagonist. We tested the hypothesis that ERα is the main receptor involved in nongenomic Ca2+ regulation. Real‐time calcium imaging showed greater decrease in [Ca2+]i with ERα specific agonist R,R THC (10nM) compared to 17βE2. TNFα (50ng/ml; 24h) upregulated ERα‐66 but downregulated ERα‐36 expression. TNFα‐induced increase in its receptor TNFRI was blunted by 17βE2. Finally, TNFα‐induced enhancement of [Ca2+]i response to histamine was attenuated by ERα activation. These data suggest that in human ASM: 1) nongenomic [Ca2+]i regulation by E2 involves ERα; b) ERα activation may be beneficial in attenuating inflammatory response to TNFα. Overall, ERs may be a novel therapeutic target in airway diseases such as asthma.

Supported by Mayo Graduate School and NIH grant UL1RR024150.
Influence of Intertukin‐13 on the Contractile Response in Mouse Airway Smooth Muscle Cells
Authors: Wijeratne Nilmini S , Perez‐Zoghbi Jose F.
Published: June 2021

Asthma is an obstructive lung disease characterized by airway hyperresponsiveness (AHR) and airway inflammation. Interleukin‐13 (IL‐13), a cytokine secreted in the airway by CD4+ cells and mast cells, plays a pivotal role in the inflammatory process. However, the role of IL‐13 in AHR is yet to be established. In this study we investigated the hypothesis that IL‐13 contributes to AHR by increasing airway smooth muscle cell (SMC) contractility. Airway contraction was investigated in mouse lung slices with phase contrast microscopy. We found that incubation of lung slices with IL‐13 (50 ng/ml) for 12 hours at 37°C increases the maximal acetylcholine‐(ACh)‐induced airway contraction as compared with slices incubated without IL‐13 (control). However, IL‐13 treatment did not alter the stimulatory concentration fifty (SC50) of the concentration‐contraction response for ACh. These results suggest that IL‐13 contributes to AHR by interacting directly with SMC to increase agonist‐induced airway contraction. Since agonist‐induced airway SMC contraction is regulated by frequency of Ca2+ oscillations and Ca2+ sensitivity, we are currently investigating the effects of IL‐13 on these parameters to understand the mechanisms of IL‐13 on airway SMCs. With this study we expect to provide some useful insights to establish the pathological mechanism of IL‐13 in asthma.
Glucagon‐like peptide‐1 prolongs cardiac action potential duration
Authors: Xiao Yong‐Fu , Nikolskaya Alena , Jaye Deborah A. , Sigg Daniel C.
Published: June 2021

Glucagon‐like peptide‐1 (GLP‐1) is a hormone predominately synthesized and secreted by intestinal L cells. The biological effects of GLP‐1 are most likely via stimulation of GLP‐1 receptors. Animal studies have shown that GLP‐1 may protect the heart from ischemic damage and improve cardiac function in heart failure. However, the effects of GLP‐1 on myocyte electrophysiology have not been investigated. In this study we assessed the effects of GLP‐1 on cardiac action potentials. Extracellular application of GLP‐1 (7‐36) at 5 nM significantly prolonged the duration of action potentials in isolated canine ventricular myocytes. The duration at 50% repolarization was prolonged from 124 ± 24 ms for control to 252 ± 30 ms for GLP‐1 (n = 6, p < 0.01). Such prolongation accompanied with an increase in L‐type Ca2+ currents. Other parameters, such as amplitude, threshold, maximum upstroke velocity of action potentials, were not changed by GLP‐1. The effect of action potential prolongation was reversible after washout of GLP‐1. Lower concentrations (<0.05 nM) of GLP‐1 had no such effect. Adding GLP‐1 (7‐36) at 30 nM to a culture medium for 24 hours significantly increased the beating rate of cultured neonatal rat ventricular cells. Our data demonstrate that GLP‐1 prolongs cardiac action potential duration and increases beating rate of cardiomyocytes. These effects probably relate to GLP‐1‐induced increase in Ca2+ currents.
Age‐related phenotypic transformation alters expression of heavy myosin isoforms SM1 and SM2 in ovine cerebral arteries
Authors: Pearce William Julian , Abrassart Jenna M , Dakanay Rina C , Williams James M
Published: June 2021

Vascular smooth muscle cells exhibit multiple stable phenotypes, including migratory, proliferative, synthetic, and contractile. The protein markers characteristic of these phenotypes are poorly defined. The present study explores the hypothesis that the heavy myosin isoforms SM1 and SM2 are reliable indicators of the extent of differentiation and contractile maturation in ovine cerebral arteries. Arteries from normoxic and chronically hypoxic (110 d at 3820 m) term fetal and non‐pregnant adult sheep were probed with antibodies against SM1 and SM2, then analyzed via Westerns and line‐scan fluorescent microscopy to determine absolute abundance of each marker as a function of relative distance from the lumen to the adventitial‐medial border. Postnatal maturation dramatically decreased the ratio of SM1 to SM2 expression approximately 2‐fold. Hypoxic acclimatization appeared to retard the rate of contractile maturation, and enhanced the SM1/SM2 ratio. Hypoxic effects were most prominent in smooth muscle near the lumen, whereas maturational effects were more evident in peri‐adventitial smooth muscle. Thus, expression patterns of SM1 and SM2 reliably reveal the age‐dependent phenotypic status of contractile smooth muscle, and appear to be influenced by independent mitogenic factors acting at the luminal and adventitial borders of ovine cerebral arteries. Supported by USPHS Grants HD31266, & HL64867.
The Contribution of Genes on Chromosome 3 to Renal Failure in the Fawn Hooded Hypertensive Rat
Authors: O'Meara Caitlin Claire , Lazar Jozef , Moreno Carol , Jacob Howard , Provoost Abraham
Published: June 2021

The fawn hooded hypertensive (FHH) rat is a well established model for hypertension and chronic kidney disease. We have performed an F2 linkage analysis between the FHH and the renal resistant and normotensive August Copenhagen Irish (ACI) strain to find quantitative trait loci (QTL) that show linkage to measures of renal impairment. Five renal failure QTL were mapped in this cross (Rf‐1 through Rf‐5), and these QTL interact with one another to produce a synergistic increase in renal impairment. To investigate the individual contribution of the Rf‐3 QTL, which is located on chromosome three, to the progression of kidney disease we have developed congenic animals that have an ACI background, and are FHH on Rf‐3, as well as Rf‐1 (rat chromosome 1) and Rf‐4 (rat chromosome 14). This triple congenic model allows us to investigate the interaction between genes in the Rf‐3 region with genes in these other two renal failure QTL. Based on proteinuria and microalbuminuria levels (figure 1) of the triple congenic animals, we have determined that genes in Rf‐3 significantly contribute to the onset and progression of kidney disease in the FHH rat. We have subsequently generated subcongenic animals targeting the Rf‐3 region to narrow the list of candidate genes, so that we can determine which specific gene(s) is contributing to observed renal impairment phenotype.
The regulation of NFAT pathway by microRNAs
Authors: Gou Deming , Weng Tingting , Zhang Honghao , Gao Li , Zhang Lisu , Liu Lin
Published: June 2021

The NFAT signaling pathway plays essential roles in organogenesis and development. MicroRNAs (miRNAs) functions in diverse biological processes. To facilitate functional analysis of miRNAs in NFAT pathway, a miRNA library overexpressing ~300 human primary miRNAs was created and used for a cell‐based high‐throughput assay with a NFAT reporter system. Fourteen miRNAs that modulate NFAT pathway have been identified. Five miRNAs (miR‐124a, 15a, 15b, 16‐1, 16‐2 and 424) down‐regulated and seven (miR‐23a, 125a, 150, 375, 26a, 26b, and 9) up‐regulated NFAT activity. Twenty‐one predicted genes were tested by their 3′‐UTR dual‐luciferase reporter assays. Nine of them were verified, including SPAG9, DYRK2, TNPO1, KPNB1, NFATc1, CAMTA1, PTBP1, CK2 and Lamc1. Three targets (NFATc1, CK2 and KPNB1) were confirmed at the protein level. PTBP1, Lamc1, and CAMTA1 were targeted by miR‐124a, and are new components in the NFAT pathway. Mutation studies revealed that the second site of the two predicted binding sites in NFATc1 was more crucial for miR‐124a‐mediated NFATc1 depression. Overexpression of miR‐124a blocked the translocation of NFATc1 protein. We propose a new mechanism of miR‐124a‐mediated NFAT regulation by targeting multiple genes. The present finding contributes towards better understanding of miRNA‐mediated NFAT regulation, which might ultimately lead to the future translation into clinical applications.
An easy method for creating your own interactive educational modules using PowerPoint
Authors: Hutchins Bob , Gray Austin
Published: June 2021

The use of interactive media by students has been shown to support the learning process when the educational tool is compatible with their needs, easy to use, and when the students perceive results. This educational approach coupled with the strain on faculty who are asked to teach more with fewer resources led us to explore how to create short, self‐paced modules using existing software, Microsoft's PowerPoint that is available to most faculty. PowerPoint offers a variety of tools to create interactive digital content. Using a combination of "trigger" and "hyperlink" functions to generate interactive buttons provided the basis for developing educational modules. Initially, we were interested in simply developing a small overview of difficult course content for students to use for their review. However, with further exploration of the versatility of Microsoft's PowerPoint software, we discovered that we could design and develop more interactive content modules that mimicked more expensive and more complex software. We chose two different areas to start this project, the complexity of the human temporal bone and the infratemporal fossa. By combining these simple interactive tools, it is suggested that any course could customize and develop interactive modules to assist students during the initial learning process and again for quick review. Variations on the temporal bone module have been in place for the past three years and the infratemporal fossa was put in place just this past year. Student evaluations indicate that they would still prefer a personal tutoring session, but they like having the ability to study on their own and at their own pace. The students also indicated that they prefer the interactive module to the text atlas. Supported by the Texas A&M Univ. Sys. HSC, Baylor College of Dentistry, Department of Biomedical Sciences
APS's Local Outreach Team in New Jersey reports on middle and high school “Neural Networks” teacher workshops
Authors: Lorentzen Laura Yvonne , Pfirrmann Cynthia , Moorman Stephen J. , Stevenson Nancy R.
Published: June 2021

With curriculum by The American Physiological Society (APS), the Local Outreach Team (LOT) in New Jersey conducted multiple workshops for science high school and middle school teachers. These workshops, entitled "Neural Networks" contained content in line with the National Science Standards and covered neurophysiology topics such as reflexes and responses. Workshops engaged the teachers (who were instructors of chemistry, biology, physics, health, or physical education) in role‐play as students, in active, inquiry‐based learning with hands‐on lab activities and associated concept mapping exercises. The Neural Networks, Active Learning Module for Middle & High School Science Classes workshop was chosen by the Society for Neuroscience (SfN) Public Education department as an exhibitor workshop at the 56th Annual National Science Teachers Association (NSTA) Conference on Science Education (2008, Boston MA). Two workshops (What is a neural network? and Neural Networks in the Body) were conducted at the 32nd Annual NJ Science Convention (2008, Somerset NJ). LOT continues to be a ready reference for the teachers as they bring the materials learned back to their own classrooms, as seen in results of evaluations and feedback surveys given to the workshop attendees. Workshop funding was provided by APS and SfN.