bluuebunny/crossref_metadata_2025 huggingface dataset:
Augmentation of Endothelium‐dependent Vasodilation during Mild Exercise Blunts Postjunctional α‐adrenergic Vasoconstriction
Authors: Kirby Brett S , Crecelius Anne R , Richards Jennifer C , Luckasen Gary J , Larson Dennis G , Dinenno Frank A
Published: June 2021

Sympathetic stimulation of postjunctional α‐adrenergic receptors elicits vasoconstriction in resting skeletal muscle but is blunted in contracting muscle in an intensity dependent manner. We tested the hypothesis that augmentation of endothelium‐dependent vasodilation during low intensity exercise attenuates α‐adrenergic vasoconstriction similarly to higher intensity exercise. We measured forearm blood flow (Doppler ultrasound) and calculated vascular conductance (FVC) to local intra‐arterial infusion of phenylephrine (PE; α1‐agonist) during mild and moderate handgrip exercise, during control non‐exercise vasodilator conditions (acetylcholine, ACH; or sodium nitroprusside, SNP), and during combined mild exercise + ACH or SNP in a total of 18 young adults. Forearm vasoconstriction to PE was observed during ACH alone and mild exercise (ΔFVC = −38±4 and −31±4%), but was blunted during moderate intensity exercise and combined mild exercise + ACH (−12±2 and −3±6%). Forearm vasoconstriction to PE was observed during SNP alone and mild intensity exercise (−36±3 and −34±4%), and was also robust during combined mild exercise + SNP (−34±6%). Constriction was again blunted during moderate intensity exercise (−15±3%). These data suggest contracting skeletal muscle may blunt postjunctional α‐adrenergic vasoconstriction through an endothelium‐dependent vasodilatory pathway.

Support NIH HL102720
Sustained ATP‐mediated vasodilation of cerebral arteries requires Ca2+ influx via TRPC3 channels to produce endothelial SKCa channel activation and hyperpolarization
Authors: Kochukov Mikhail Y , Abramowitz Joel , Birnbaumer Lutz , Marrelli Sean P
Published: June 2021

Extracellular ATP acts through endothelial cell (EC) purinoceptors to produce vasodilation of cerebral arteries (CA). IKCa channels have been established in this mechanism; however, the role of SKCa channels is not clear. We hypothesize that sustained ATP‐mediated hyperpolarization and vasodilation requires Ca2+ influx via EC TRPC3 channels to produce activation of SKCa channels.

Methods

Vascular diameter was measured following luminal delivery of ATP in mouse pressurized superior cerebellar arteries (SCA). Changes in EC Ca2+ were measured by biosensor in SCA isolated from Cx40BAC‐GCaMP2 transgenic mice. Membrane currents and voltage were measured in freshly isolated EC from CA of WT and TRPC3 knock out (KO) mice using perforated patch whole cell recording in voltage or current clamp mode.

Results

Application of a selective TRPC3 blocker (Pyr3) significantly attenuated the duration of endothelial Ca2+ increase and vasodilation to ATP in pressurized SCA and abbreviated ATP‐mediated hyperpolarization in freshly isolated ECs. Responses were replicated with arteries and EC isolated from TRPC3 KO mice. Selective SKCa channel inhibition (UCL1684) mirrored the abbreviated time course of EC hyperpolarization, suggesting a common pathway.

Conclusion

We propose that Ca2+ influx via TRPC3 promotes SKCa activation to produce the sustained component of cerebral vasorelaxation to ATP. Supported by R01HL088435 to SPM
Effect of aging on carotid baroreflex function during muscle mechanoreflex activation with and without concurrent metabolite accumulation in humans
Authors: Drew Rachel C , Muller Matthew D , Blaha Cheryl A , Mast Jessica L , Herr Michael D , Sinoway Lawrence I
Published: June 2021

In humans, the muscle mechanoreflex evoked by stretch resets carotid baroreflex (CBR) control of heart rate (HR) and mean arterial pressure (MAP) to higher levels with and without concurrent metabolic input (Drew et al., 2008). The effect of aging on these responses is unknown, prompting us to examine its effect on CBR‐HR and CBR‐MAP control during mechanoreflex activation with and without concurrent metabolic input in humans. 12 young (Y; 27±1 y) and 6 old (O; 63±1 y) subjects underwent 3 min of calf stretch during circulatory occlusion (CO). In a separate trial, stretch was preceded by 1.5 min of isometric calf exercise and CO, which trapped metabolites within the calf circulation. Maximal gain (GMAX) and operating point gain (GOP) of CBR‐HR were lower in O compared to Y at rest (p<0.05). GMAX and GOP were also lower in O compared to Y during stretch (Y; −0.45±0.11 vs. O; −0.17±0.04 and Y; −0.32±0.10 vs. O; −0.05±0.01 b.min−1.mmHg−1, respectively; p<0.05), as well as stretch with metabolic input (p<0.05). Stretch reset CBR‐HR and CBR‐MAP curves to higher HRs and MAPs similarly in both groups, both with and without metabolic input. In conclusion, the muscle mechanoreflex resets CBR control of HR and MAP to higher levels in Y and O humans. CBR‐HR control is less sensitive in O than Y humans at rest, as well as during muscle mechanoreflex with and without concurrent metabolic input. Supported by P01 HL096570 (LIS); UL1 TR000127 (LIS).
Endurance training fails to correct age‐related declines in cardiovagal control
Authors: Sawicki Carolyn , Luchyshyn Torri A , Frances Maria F , Norton Katelyn N , Shoemaker J Kevin
Published: June 2021

This study tested the hypothesis that long‐term endurance training (ET) preserves cardiovagal control in aging individuals. Baroreflex sensitivity (BRS) and the rapid heart rate response at exercise onset (ΔHR) reflect levels of cardiovagal control. BRS declines with age and cardiovascular disease but, reportedly, this effect can be reversed by ET. It remains unclear if ET also preserves ΔHR (reflecting removal of parasympathetic cardiac inhibition). Both BRS (sequence method, 5–10 min of baseline) and ΔHR to three, 30s contractions at 40% of their maximum voluntary contraction strength, were assessed in a group of young individuals (Y; age=26±4) and in groups of older individuals who were healthy (O; age=56±4), endurance trained (ET; age=55±4) or entering cardiac rehabilitation (CR; age=59±4) (n=15 for each group). Compared with Y (10±9 bpm), ΔHR was less in CR (3±2 bpm; P<0.05) but not the other groups (O: 7±5, ET: 5±3 bpm; NS). Similarly, compared with Y (30±15 ms/mmHg), BRS was less in CR (14±12 ms/mmHg) but not the other groups (O: 20±12, ET: 25±15 ms/mmHg). Contrary to previous results, ET did not preserve BRS at levels expressed in Y. However, age correlated strongly with BRS and ΔHR (r=−0.4, p=0.001; r=−0.3, p=0.013 respectively) across all groups. The results suggest that age exerts a dominant impact on cardiovagal control and that this effect remains difficult to restore with long‐term ET. Supported by CIHR.
Network interactions within the intrinsic cardiac nervous system: Implications for reflex control of regional cardiac function
Authors: Beaumont Eric , Armour John Andrew , Salavatian Siamak , Vinet Alain , Jacquemet Vincent , Ardell Jeffrey L
Published: June 2021

Objective

To determine how aggregates of intrinsic cardiac (IC) neurons transduce the cardiovascular milieu vs respond to changes in central drive. Also to determine how IC network interactions, subsequent to induced neural imbalances, subserve the genesis of atrial fibrillation (AF).

Methods

Activity from multiple IC neurons within the right atrial ganglionated plexus was recorded from anesthetized canines. Induced changes in IC neuronal activity were evaluated in response to: (1) cardiac touch; (2) electrical activation of the cervical vagus or stellate ganglia; (3) occlusion of the inferior vena cava or thoracic aorta; (4) focal left ventricular ischemia and (5) neurally induced atrial arrhythmias (AF).

Results

The majority of IC neurons were local circuit in nature and in basal states displayed low level functional interconnectivity. The majority of IC neurons received indirect central inputs (vagus and stellate) and a lesser proportion transduced the cardiac milieu including responding to multimodal stressors applied to the great vessels and heart. In response to mediastinal nerve stimulation most IC neurons became excessive excited and their functional interconnectivity enhanced; such network behavior preceding and continuing throughout AF.

Conclusion

Stochastic interactions among IC neuronal populations underlie control of regional cardiac function. (Supported by HL71830)
Epi/pericellular protein disulfide isomerase operates a redox‐dependent pathway sustaining vascular caliber after mechanical injury
Authors: Tanaka Leonardo Yuji , Hironaka Gustavo Ken , Araújo Haniel Alves , Takimura Celso Kiyochi , Laurindo Francisco Rafael Martins
Published: June 2021

The endoplasmic reticulum (ER) chaperone Protein Disulfide Isomerase (PDI), also present at the cell surface (epi/pericellular = epcPDI), is involved in redox pathways. We tested whether PDI regulates vascular repair after injury (AI). After rabbit iliac artery balloon injury, there was marked increase in PDI mRNA and protein 4, 7 and 14 days AI vs. basal, with increased intracellular and epcPDI protein expression. Inhibition of epcPDI with neutralizing antibody (AbPDI) in vivo (perivascular gel delivery) from days 12–14 AI decreased ROS signals and nitrogen oxide levels. Importantly, AbPDI decreased vascular caliber area vs. control Ab (2.31±0.68 vs. 4.01±0.73 mm2, p<0.05, intravascular optical coherence tomography), with parallel decrease in total vessel area, but no change in wall area. EpcPDI silencing effects involve disruption of collagen matrix and actin cytoskeleton, with lower expression of collagens I and III. The capacity to sustain isometric tension was also decreased. PDI knockdown with siRNA (in vitro organ culture) 14 days AI enhanced ER stress, cell death, proliferation and loss of smooth muscle differentiation markers. Thus, PDI is strongly expressed AI; intracellular PDI sustains stress adaptation and favors anti‐neointimal processes, while epcPDI acts against contrictive remodeling through redox mechanisms linked to matrix/cytoskeletal organization. (Support: FAPESP, CNPq)
Cardiomyocyte specific overexpression of C3orf58 activates ER stress leading to impaired cardiac function
Authors: Zhang Zhiping , Mirotsou Maria , Payne Alan , Mu Hui , Zhang Lunan , Finch Elizabeth A , Pratt Richard E , Rosenberg Paul , Dzau Victor J
Published: June 2021

Although accumulating evidence supports the crucial role of ER stress in cardiovascular diseases, essential information is still required for its role in the pathogenesis of these disorders. We previously reported the identification of C3orf58 (aka HASF) as a novel stem cell derived factor involved in cytoprotection. Transgenic mice with a high‐level of cardiac specific overexpression of HASF showed reduced fractional shortening as early as 4 weeks old. Neonatal cardiomyocytes isolated from these mice showed reduction in basal Ca2+ levels, abnormal calcium oscillation and a dramatic reduction in magnitude of store‐operated Ca2+ entry after stimulation. Adult transgenic cardiomyocytes exhibited reduced L type channel currents. Notable, all three pathways involved in ER stress, PERK, ATF6 and IRE1, were activated in the hearts of transgenic mice as early as 2 weeks old. Moreover, to demonstrate the specific effect of HASF on ER stress, fibroblasts isolated from HASF heterologous KO were treated with ER stress inducers thapsigargin and tunicamycin. The induction of ER stress sensor GPR78 was significantly lower in these treated fibroblasts. Based on our findings we propose that C3orf58 is a novel regulator of ER stress pathways whose overexpression is involved in cardiac pathology and disease.

The study was supported by NHLBI/NIH grants RO1 HL35610, HL81744, HL72010, HL73219 and Foundation Leducq (to VJD).
Isoflurane (ISO) Inhibits Bronchopulmonary C‐fiber‐mediated Apneic Response to phenylbiguanide (PBG) by Depressing 5‐HT3 Receptor Function in Rats
Authors: Zhang Zhenxiong , Zhuang Jianguo , Zhang Cancan , Xu Fadi
Published: June 2021

Our previous study has shown that isoflurane (ISO), a commonly used volatile anesthetic, has an excitatory effect on bronchopulmonary C‐fibers (PCFs) (Zhang, et al., EB 2011, A377). Because right atrial bolus injection of PBG has been extensively used to selectively stimulate PCFs by action on local 5‐HT3 receptor, and thereby evoking an apnea, we asked whether inhalation of ISO would facilitate the PCF 5‐HT3 receptor‐mediated apneic response, and if so, how. Our experiments were performed in three groups of rats. We recorded the ventilatory response and pulmonary C neural response to right atrial bolus injection of PBG (25 μg/kg) before and during inhalation of 5% ISO. To this end, the ventilatory response was recorded in anesthetized and spontaneously breathing rats (Group I), while pulmonary C neurons were electrophysiologically isolated and extracellularly recorded in the nodose ganglion of anesthetized and paralyzed rats (Group II). Using perforated patch clamp technique in vitro, we further tested the effect of ISO (1 mM) on the PBG‐induced inward current of pulmonary C neurons labeled by previously instilling 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI) into the airways and lungs (Group III). Our results showed that inhalation of ISO markedly shortened the PBG‐produced apnea and attenuated the PBG‐elicited excitation of pulmonary C neurons. Moreover, ISO perfusion depressed the PBG‐induced inward current of pulmonary C neurons. These data suggest that ISO can inhibit PCF 5‐HT3 channel function to attenuate PCF excitatory response to PBG, likely contributing to shortening the PBG‐induced apnea by ISO in rats. (Supported by HL 107462 and ALA RG‐191095‐N)
Changes in renal sympathetic nerve activity during and after exposure to acute intermittent hypercapnia
Authors: Valic Zoran , Pecotic Renata , Dogas Zoran , Valic Maja
Published: June 2021

Elevated levels of carbon dioxide (hypercapnia) and/or decreased level of oxygen (hypoxia) act through neural networks to influence ventilatory and cardiovascular function. Both hypercapnic and hypoxic stimuli can evoke increase in sympathetic nerve activity and it appears that hypoxia can evoke prolonged duration of this increase. The present study was performed to investigate the renal sympathetic nerve activity in urethane‐anesthetized, mechanically ventilated Sprague‐Dawley rats exposed to five hypercapnic stimuli each lasting 3 minutes. Blood pressure and renal sympathetic nerve activity were measured and recorded throughout the experiment. Analysis was performed in the period before and during hypercapnic exposures (15% of the CO2 in the air), as well as 15, 30 and 60 minutes after the last hypercapnic episode. Renal sympathetic nerve activity increased to 190.3±22.8% during hypercapnia when compared to baseline. At 15 minutes after the last hypercapnic exposure nerve activity remained elevated at 138.0±14.8%. At 30 and 60 minutes renal sympathetic nerve activity was 136.8±21.5% and 131.8±18.3%, respectively. Acute intermittent hypercapnia evoked increase in renal sympathetic nerve activity that lasted at least 60 minutes after cessation of stimulus.

This study was supported by Croatian Science Foundation.
The effect of sleep restriction on the respiratory and vascular control
Authors: Pugh Keith , Taheri Shahrad , Balanos George
Published: June 2021

Total sleep deprivation is known to disrupt respiratory and vascular control. However, partial sleep deprivation is far more common and its effects less known.

Participants (mean age ± S.D = 23.4 ± 1.5 years) with normal habitual sleeping patterns undertook two protocols. A sleep restriction protocol consisted of a baseline night of 8 hours sleep, followed by 3 nights of 4 hours sleep. A control protocol consisted of the same initial night followed by 3 nights of 10 hours of sleep opportunity. Chemosensitivity and endothelial function measurements were performed each day.

Central chemosensitivity increased by 51.6 ± 9.4 % after the second night of sleep restriction but returned to baseline after the third night. In the control protocol central chemosensitivity remained unchanged. Peripheral chemosensitivity did not differ between the two protocols. Endothelial function measured by flow‐mediated dilation was decreased by 46.7 ± 1.6 % after the second night of sleep restriction but recovered after the third night. No changes were observed in endothelial function during the control protocol.

These results suggest that acute sleep restriction affects both respiratory and vascular control with a complex time course. It is plausible that partial sleep deprivation can be a precursor to conditions such as sleep apnoea and cardiovascular disease.
Progesterone enhances respiratory frequency and reduces apnea frequency during sleep in mice KO for the nuclear progesterone receptor
Authors: Marcouiller François , Lavoie Raphael , Joseph Vincent
Published: June 2021

We used adult female mice knocked‐out for the nuclear progesterone receptor gene (PRKO) and wild‐type (WT) controls to record breathing pattern and apnea frequency across sleep/wake states (determined by EEG/EMG analysis). Respiratory frequency (fR) was similar in WT and PRKO mice during quiet wakefulness. During nREM and REM sleep, fR declined respectively by 31 ± 3 % and 27 ± 5 % in PRKO mice and by 17 ± 7 % and 11 ± 15 % in WT mice (p<0.0001 sleep state effect: p=0.03 group effect). Apnea frequency during sleep (nREM+REM) was 13.8 ± 6 h−1 in WT and 30.4 ± 18 h−1 in PRKO mice. A group of PRKO female mice was treated with progesterone for 7 days (eq. 4mg/kg/day ‐ subcutaneous pump), and compared to the PRKO and WT females treated with vehicle. After progesterone treatment in PRKO mice, fR values were 12 ± 5 % and 7 ± 5 % lower during nREM and REM sleep compared to wakefulness (no significant sleep state effect). Apnea frequency during sleep in PRKO mice treated with progesterone was 2.8 ± 2 h−1, markedly lower than in PRKO mice treated with vehicle. We conclude that deletion of the nuclear progesterone receptor aggravates the respiratory depression during sleep and enhances apnea frequency. Since progesterone treatment in PRKO mice enhances fR and reduces apnea frequency during sleep, progesterone also acts on the respiratory control system through other signalling pathways. Founded by CIHR.
The role of ATP and glia in modulating the generation of sighs
Authors: Dashevskiy Tatiana , Doi Atsushi , Ramirez Jan‐Marino
Published: June 2021

The Pre‐Bötzinger Complex (PreBötC), a medullary brainstem region, is essential for generating breathing. Isolated in a transverse slice preparation, the preBötC continues to generate under normal conditions “fictive eupneic activity” and at a lower frequency also “fictive sighs”. The mechanisms generating the periodicity of fictive sighs as well as its intrinsic nature are largely unknown. Here, we explored the role of ATP in sigh generation. Activation of P2Y receptors increased fictive sigh frequency, while activation of P2X receptors had no significant effect on sigh frequency. Moreover, blockade of P2Y1 receptors abolished fictive sighs during the first 20–30 min of drug bath application. These in vitro results were also confirmed in adult in vivo mice. We hypothesized that the release of ATP and activation of P2Y receptors is coupled to the increase of astrocytic intracellular Ca2+. Indeed, bath application of glial toxin, fluorocitric (FC) acid, abolished sighs but not eupneic activity. The neuromodulators 5‐HT, DOI, norepinephrine or isoproterenol that induce fictive sighs under normal conditions fail to induce fictive sighs in the presence of FC acid. We conclude that astrocytic ATP may play a critical role in modulating sigh generation. In addition, glia may also be involved in the hypoxic response since the hypoxia‐induced augmentation of eupneic activity was abolished in the presence of FC acid.
Quantification of enkephalinergic immunoreactivity in the periaqueductal gray of an animal model of endometriosis
Authors: Torres‐Reveron Annelyn , Cruz Myrella L , Hernandez Siomara , Hernandez Anixa , Appleyard Caroline B.
Published: June 2021

Endometriosis is a debilitating disease characterized by pelvic pain, dysmenorrhea and dyspareunia. These symptoms produce stress on the individual and impact daily life activities. It is still poorly understood how stress affects endometriosis associated pain. We aimed to measure levels of the endogenous opioid peptide enkephalin (ENK) in the periaqueductal gray (PAG) of an animal model of endometriosis since PAG plays a critical role in descending modulation of pain via opioidergic mechanisms. Four groups of female rats received either: sham surgery, or surgical endometriosis then no‐stress, controllable or uncontrollable swim stress during 10 consecutive days. Sixty days after surgery, rats were killed and perfused. The brains were removed and sections containing the PAG processed for quantitative light immunohistochemistry for ENK. Results showed that uncontrollable stress exacerbated the vesicle grade in animals with endometriosis as compared to no‐stress or controllable stress. Within the PAG, ENK was mostly observed in the dorsal and lateral regions thru the rostral‐caudal span. Optical densitometry quantification revealed no differences between groups in ENK levels suggesting that neither stress nor endometriosis is affecting ENK levels in the PAG. Future experiments will elucidate whether other opioid peptides or their receptors alter in response to stress and endometriosis. R15AT006373
Nicotinic Acetylcholine Receptors expressed by Muscle Afferent Neurons
Authors: Baxter James Clifford , Elmslie Keith S
Published: June 2021

Background

The Exercise Pressor Reflex (EPR) is activated during exercise to increase cardiovascular function. Acetylcholine (ACh) triggers muscle contraction and could be a potent signal of muscle activity during exercise. We show that nicotinic ACh receptors (nAChR) are expressed by muscle afferents.

Methods

Identified muscle afferent neurons were isolated from male Sprague‐Dawley rats. ACh‐induced currents were measured using the patch clamp technique. All neurons had diameters < 40 μm and expressed Nav1.8 channels. The role of α7 nAChR was tested using α‐Conotoxin IMI.

Results

ACh‐induced current was measured in 48% (22/45) of muscle afferents. Of the ACh responsive neurons, 9 responded to capsaicin and 9 responded to ATP. 63% (7/11) of ACh responses were blocked by α‐Conotoxin IMI (α7). We are also testing blockers of other nAChRs.

Conclusion

nAChRs are expressed by muscle afferent neurons and α7 nAChR mediate the majority of these responses. It will be interesting to test α7 blockers in vivo to determine the role of nAChRs in the EPR. nAChRs could be pharmacological targets for suppressing the EPR in patients with peripheral vascular disease.
Oral administration of high doses of pseudoephedrine (PSE) hydrochloride accelerates the onset of CNS oxygen toxicity (CNS‐OT) seizures in rats exposed to hyperbaric oxygen (HBO) at 5 atmospheres absolute (ATA)
Authors: Pilla Raffaele , Held Heather E. , Landon Carol , Dean Jay B.
Published: June 2021

Commonly, PSE salts are utilized by scuba divers as nasal decongestants. Anecdotal reports from Divers Alert Network (DAN) suggest that taking PSE prior to a deep dive while breathing pure O2 (i.e., Nitrox or using a rebreather) increases the risk for CNS‐OT. We hypothesized that high doses of PSE reduce latency to seizure (LS) in male and female Sprague‐Dawley rats breathing a lethal dose of HBO (5ATA).

63 male and 63 female rats were implanted with radio‐transmitters for recording EEG, body temperature (BT) and activity. After ≥7 days, and 2hr before “diving”, rats were orally administered either saline solution (control) or PSE hydrochloride at the doses of (mg PSE/Kg): 0, 40, 80, 100, 120, 160 & 320 (LD50for PSE in rats is 640 mg/Kg). Rats were exposed to pure O2 at 5ATA until the onset seizures and LS was measured.

We observed a significant decrease in LS at doses of 80 and 320 mg/Kg in females, whereas in males data displayed a more regular dose‐dependent decrease, notably at dosages of 100–320 mg/Kg. No consistent variation was observed at other doses. In addition, BT consistently dropped after the administration of each PSE dose, highlighting direct correlations among PSE dose, BT and LS (females).

High doses of PSE are potentially harmful breathing pure O2 at 5 ATA of depth. Doses taken by commercial, technical and military divers breathing oxygen‐enriched gas mixes should not exceed the recommended guidelines. (DAN)
REPEATED ACUTE RESTRAINT‐INDUCED STRESS: EFFECTS ON BODY WEIGHT, CORTISOL AND REPRODUCTIVE HORMONES IN FEMALE WISTAR RATS
Authors: Olayaki Luqman Aribidesi , Suleiman Sheu Oluwadare
Published: June 2021

Stress through its response, be it chronic or acute, produces behavioural and chemical deficits that affect most body functions especially reproduction. Although chronic exposure to stress has been reported to affect reproduction in an adverse manner, effects of acute exposure to stress on reproduction have been controversial. To study the effects of repeated acute restraint‐induced exposure to stress on reproductive hormones in female rats, fifteen (15) female Wistar rats were divided into three groups of five (5) rats each. Groups I and II rats were restrained for 10 minutes and 20 minutes respectively for seven (7) days while group III rats served as control. All the rats were weighed everyday before the restraint throughout the duration of the experiment. On the 7th day, immediately after the restraint, the rats were sacrificed and their blood samples collected into heparinized tubes and centrifuged. The supernatant plasma from each blood sample was then used to analyse reproductive hormones and cortisol. The results showed that there were increases in weights and hormonal levels of the restrained rats compared to the control. Observation of daily average weights indicated weight increase from day 2 to day 7. In 10 minute restrained rats, levels of oestradiol (240 ± 17.55pg/ml), progesterone (41.25 ± 1.94ng/ml) and FSH (1.33 ± 0.06mIu/ml) significantly increased when compared to their corresponding control values of (174.00 ± 8.28pg/ml), (13.13 ± 1.40ng/ml) and (0.3 ± 0.3mIu/ml) respectively. Levels of cortisol (7.63 ± 0.76μg/ml), LH (4.80 ± 0.49mIu/ml) and prolactin (4.25 ± 0.32ng/ml) also increased in the 20 minute restrained rats compared to their control values of (3.90 ± 1.10μg/ml), (4.42 ± 0.28mIu/ml) and (3.50 ± 0.20ng/ml) respectively but the increases were not significant. Thus, it was concluded that acute stress such as 10‐minute and 20‐minute restraint used in this study could lead to increases in body weight and reproductive hormones.
Cadmium Transport in Epithelial Cells of a Crab: comparison between Gills and Hepatopancreas
Authors: Zanotto Flavia Pinheiro , Ortega Priscila , Castro Julia Mourão , Santos Rudney Almeida
Published: June 2021

Cadmium (Cd) is a non‐essential metal for crustaceans and is responsible for alterations in ion transport in a number of animals. Cd transport has not been studied in detail in crustaceans. Therefore, the present work was undertaken to characterize Cd transport in gill and hepatopancreatic cells of a crab, Ucides cordatus, found in contaminated mangrove regions. Cells were marked with a fluorescent probe (Fluo‐3) and the change in fluorescence was followed with the addition of Cd (0, 1.0 and 1.5 ìM), ATP (1.5 ìM) and different inhibitors. Results show that Cd enters gill cells and the fluorescence decreases to baseline values within few seconds. Cd transport for posterior gills is double that for anterior gills in terms of fluorescence change (1,600 against 800 ÄF × 5s−1 × 22.104 cells, respectively). ATP added together with Cd at 1.5 ìM causes a decrease in fluorescence, suggesting an efflux of intracellular Cd. For hepatopancreas, different cell types display different Cd uptake. Embryonic (E) and resorptive cells (R) showed the highest Cd uptake compared to the other cells, and the uptake was lower than that seen for gill cells (1,600 against 1,000 ÄF × 5s−1 × 22.104 cells, respectively). Results show that Cd enters gill and hepatopancreatic cells, and equilibrates intracellularly within 4–5 seconds. Moreover, Cd influx is followed by a rapid cell sequestration and a decrease of intracellular Cd in these crab cells.
Morpholino oligonucleotide knockdown reveals extracellular calcium‐sensing receptor role in zebrafish skeletal development
Authors: Herberger Amanda L , Loretz Christopher A
Published: June 2021

The complex vertebrate skeleton depends on regulated cell activities to lay down protein matrix and mineral components. Bone, a distinctive vertebrate characteristic, is a storage site for physiologically‐important calcium ion. The extracellular calcium‐sensing receptor (CaSR), a G protein‐coupled receptor that is tightly linked to homeostatic regulation of calcium by endocrine glands, ion‐transporting epithelia and bone, is restricted to the chordate‐vertebrate evolutionary lineage. Our overall research objective is to understand the functional linkage of CaSRs to skeleton in the context of vertebrate evolution in diverse habitats. Since little is known about CaSR in skeletal biology of non‐mammalian vertebrates, immunohistochemistry was applied to adult and embryonic zebrafish, revealing CaSR protein expression in many tissues, including chondrocytes and notochordal sheath cells. Morpholino oligonucleotide (MO) knockdown was used to discover CaSR role(s) in this zebrafish model system. By RT‐PCR, MO treatment reduced CaSR mRNA expression measured at 1 and 2 dpf in TU strain fish. Corresponding to the MO knockdown of normally‐spliced CaSR mRNA, we observed a phenotype characterized by stunted growth and disorganization of the notochordal component of axial skeleton by 1 dpf. We conclude that, like to its essential role in bone development in mammals, CaSR is important in skeletogenesis in fish.
RNA‐seq reveals a robust transcriptomic response during anoxia in the Western painted turtle
Authors: Warren Daniel E. , Hill Craig A. , Kandoth Cyriac , Buck Leslie T.
Published: June 2021

Western painted turtles, Chrysemys picta bellii, can survive 3 months of anoxia at 3°C and for more than 30 hours at 20°C. Although the physiological responses to anoxia have been well studied, the underlying transcriptomic responses are not as well‐characterized. Using RNA‐seq methodology, we show a robust transcriptional response in ventricle and telencephalon to 24 hours of anoxia at 19°C, including the upregulation of several genes by more than 100‐fold, despite decreases in total RNA content of 50% and 12% in ventricle and telencephalon, respectively. qPCR validated the increased expression of several genes in ventricle, including FOS (91‐fold) and a GLUT1‐like transporter belonging to SLC2A (29‐fold). Similar validations were made in telencephalon for APOLD1 (292‐fold) and PTGS2 (219‐fold). There were no decreases in telencephalic gene expression, but there were decreases in 5 ventricular genes, including CDO1, which, when knocked out in mice, increases hydrogen sulfide levels, SRSF5, a gene involved in mRNA processing, and MKNK1, which regulates growth and differentiation. This study shows how the increased availability of sequenced genomes can provide an unprecedented level of insight into our understanding of extreme physiological adaptation and marks a transformative moment in comparative physiology. This study was funded by Saint Louis University (DEW) and NSERC (LTB).
Prolonged food deprivation increases mRNA and protein expression of deiodinase 1 & 2, and thyroid hormone receptor β‐1 in a fasting‐adapted mammal
Authors: Martinez Bridget , Sonanez Jose Guadalupe , Ortiz Rudy M
Published: June 2021

Food deprivation in mammals is typically associated with reduced [thyroid hormone] (TH), and deiodinase content and activity to suppress metabolism, but in hibernating, metabolically quiescent ground squirrels, [TH] increase in the presence of reduced TH receptor content and activity making them cryptically hyperthyroid. However, in prolong‐fasted, metabolically active elephant seal pups, TH levels are maintained, if not elevated. The functional relevance of this apparent paradox is unknown and clearly demonstrates variability in the regulation of [TH] and function in food‐deprived mammals. To address the hypothesis that prolonged fasting (7 weeks) increases the expression of deiodinases (DI1/2) and TH receptor (THrβ‐1), we measured the mRNA expression of these genes in adipose and muscle at 1, 3 and 7 wks fasting. Fasting did not decrease plasma [thyroid stimulating hormone] (TSH), total [tri‐iodothyronine] (tT3), free [T3] (fT3), total [thyroxine] ( tT4) and free [T4] (fT4) suggesting that the hypothalamic‐pituitary‐thyroid (HPT) axis is maintained during fasting. The mRNA and protein (except adipose DI1) expressions of adipose and muscle DI1 & 2 increased suggesting that the mechanisms mediating cellular TH activity are increased with prolonged fasting. Fasting also increased adipose and muscle THrβ‐1 mRNA expression. The data demonstrate a unique, atypical mechanism of TH activity and regulation in mammals adapted to prolonged food deprivation in which the potential responsiveness of peripheral tissues and cellular TH activity are increased, likely to meet the energetic demands of prolonged fasting. This unique cellular response may contribute to the seal's adaptation to prolonged fasting, providing insight to therapies for TH‐related pathologies in humans.
Intermittent Hypoxia‐Induced Insulin Resistance Is Ameliorated By Endothelin‐1 Receptor Type B Antagonist
Authors: Polak Jan , Punjabi Naresh M. , Shimoda Larissa A.
Published: June 2021

Obstructive sleep apnea (OSA) is associated with decreased insulin sensitivity (IS). Endothelin‐1 (ET‐1) impairs insulin signaling and alters glucose homeostasis. Patients with OSA and mice exposed to intermittent hypoxia (IH) have elevated ET‐1 levels. We examined whether blockade of ET‐1 receptors type A (ETA) and type B (ETB) ameliorates worsening of IS under IH in mice. Subcutaneously implanted pumps delivered BQ‐123 (ETA antagonist; 200 nmol/kg/d), BQ‐788 (ETB antagonist; 200 nmol/kg/d) or vehicle (saline or propyleneglycol [PG]) for 14 days in C57BL6/J mice (10/group). During treatment, mice were exposed to IH or intermittent air (IA). IH was induced by decreasing the FiO2 from 20.9% to 6% (60/hr). After IH or IA exposure, insulin was injected intraperitoneally (0.5 IU/kg) and plasma glucose determined at 0, 10, 20, 30, 40, 50, 60, 90 and 120 minutes after injection. IS was assessed as the area under glucose curve (AUC). 14‐days of IH impaired IS (AUCglucose: 7185±401 vs 8699±401; p<0.05). Treatment with BQ‐788 improved IS under IA (AUCglucose: 5281±401, p<0.05) and prevented worsening of IS with IH (AUCglucose: 7302±401, p<0.05). While BQ‐123 treatment also improved IS under IA (AUCglucose: 6557±545, p=0.05), no effect was observed under IH (AUC: 9989±599, p>;0.05). Treatment with selective ETB receptor antagonist prevented worsening of IS with IH, suggesting that ET‐1 plays a role in glucose homeostasis.
Regulation of skeletal muscle strength by estradiol: neuronal nitric oxide synthase and myosin regulatory light chain
Authors: Lai Shaojuan , Lowe Dawn A.
Published: June 2021

Loss of skeletal muscle strength is a significant health concern with aging. In women, increased loss of strength happens during menopause, suggesting that the loss is related to changes in ovarian hormones. Estradiol, a predominant hormone secreted by ovaries, plays an important role in the function of the key contractile protein, myosin, and thus muscle strength in young adult, female mice. However, the mechanism(s) by which estradiol affects strength in females is not known. The goal of this study is to elucidate the mechanism of estradiol on musculoskeletal strength in female mice. We found the expression of neuronal nitric oxide synthase (nNOS) and the phosphorylation of myosin regulatory light chain (RLC) in muscles of ovariectomized mice were reduced 52–74%, compared to ovary intact‐mice (P<0.001). Estradiol treatment reversed the expression of nNOS and phosphorylation of RLC. In addition, reduced nNOS activity resulted in decreased phosphorylation of RLC. Using a complementary C2C12 cell culture approach, we found that these effects are mediated by estrogen receptor beta (ERß). These results indicate that estradiol regulates skeletal muscle nNOS expression and RLC phosphorylation through ERß, which in turn may contribute to changes in muscle strength in females.
Effect of Trenbolone enanthate on protein degradation in levator ani/bulbocavernosus (LABC) muscle in orchiectomized rats
Authors: Ye Fan , McCoy Sean C , Yarrow Joshua F , Conover Christine F , Beck Darren T , Beggs Luke A , Beharry Adam W , Judge Andrew R , Ross Heather H , Borsa Paul A , Tillman Mark D , Conrad Bryan P , Pingel Jennifer E , Wronski Thomas J , Johnson Sally E , Kristinsson Hordur G , Borst Stephen E
Published: June 2021

Trenbolone (TREN), a potent testosterone analog, does not undergo 5[alpha]‐reduction to the more potent metabolite dihydrotestosterone (DHT) and may produce androgenic effects without DHT‐mediated adverse effects. The purpose of this study was to investigate the mechanism underlying the trenbolone – induced augmentation of LABC muscle mass in orchiectomized (ORX) rats. Forty, 10 month old male F344/Brown Norway rats were randomized into SHAM, ORX, ORX+TE, and ORX+TREN groups. Following surgery, animals recovered for 1 week and then were treated with vehicle, TE, or TREN by weekly i.m. injection. 5 weeks of TE administration nearly doubled prostate mass, while TREN resulted in a nonsigificant reduction in prostate mass, compared with SHAMs. ORX induced a significant 2‐fold elevation atrogin‐1 and a 3‐fold elevation in Muscle RING Finger‐1 mRNA expression, effects which were completely abolished by TE and TREN. Interestingly, TREN induced a greater decrease in atrogin‐1 gene expression compared to TE. These data suggest that TREN may prevent muscle loss after ORX through inhibiting master genes that regulate skeletal muscle atrophy. This work was supported by VA Merit Award to SEB and a VA Career Development Award‐2 to JFY.
Role of Trans‐signaling Molecules from Lewis Lung Carcinoma (LLC) Cells on C2C12 Myotube Atrophy and Protein Turnover Regulation
Authors: Gao Song , Carson James A.
Published: June 2021

Cancer cachexia is a metabolic wasting syndrome observed in many cancer patients that is characterized by unintentional body weight loss. Imbalances in rates of protein synthesis and degradation are responsible for cancer‐induced skeletal muscle wasting. Lewis lung carcinoma (LLC) cell implantation is a widely used explant mouse model for examining cancer cachexia and muscle wasting. The purpose of this study is to investigate the influence of trans‐signals induced by LLC tumor cells on C2C12 myotube morphology and protein turnover regulation. C2C12 myotubes were treated with LLC cell culture medium (0%, 15% and 25%) for 4h, 24h and 72h. LLC medium treatment (25% for 72h) significantly decreased myotube diameter. The LLC medium induced both Stat3 and AMPK phosphorylation, while decreasing mTOR (S2448) and 4EBP1 (T37/46) phosphorylation in dose dependent manner. In addition, LLC treatment activated the FOXO3/Atrogin‐1 protein degradation signaling pathway. These results demonstrate that trans‐signaling molecules from LLC tumor cells can result in C2C12 myotube atrophy by both inhibiting mTOR regulation of protein synthesis pathway and activating protein degradation. (RO1CA121249)
High lipid concentrations regulate skeletal muscle REDD1
Authors: Willliamson David L , Dungan Cory M , Jadhav Kavita S
Published: June 2021

Upregulation of REDD1 expression, due to glucocorticoids, diabetes, or hypoxia, has been reported in skeletal muscle. Sharing similar regulators, REDD1s role in obese skeletal muscle growth has not been established. The objective of these studies was to examine the effects of high in vivo and in vitro lipid concentrations on REDD1 and downstream targets in skeletal muscle. Plantar flexor muscle from lean and ob/ob mice and mice fed a low (10%) or a high (60%) fat diet for 8 wks, and C2C12 myoblast cultures treated with 50μM C2‐ceramide or a control were analyzed for protein expression of REDD1 and Akt/mTOR activation. The high fat diet increased (p<.05) fasting REDD1 muscle expression versus the low fat group, which was associated with aberrant mTOR signaling. Fasted ob/ob mouse (vs lean) muscle phosphorylation of rpS6 was elevated and Akt repressed (p<.05). Unlike the lean (p<.05), ob/ob mice had a limited increase in rpS6 and Akt phosphorylation to feeding, and higher REDD1 protein expression (p<.05; vs lean) regardless of nutrient status. REDD1 was elevated (p<.05; vs controls) in ceramide treated, insulin‐insensitive myoblasts, which was associated with reduced Akt and rpS6 phosphorylation. Our results indicate that high lipid concentrations upregulate REDD1 expression, causing aberrant responses in growth signaling activation. Support: University at Buffalo Start Up Funds
GLUTAMATE AND NITRIC OXIDE INTERACTION IN THE ROSTROVENTROLATERAL MEDULLA (RVLM) OF WISTAR RATS SUBMITTED TO EXERCISE TRAINING
Authors: Martins‐Pinge Marli , de Ataides Raquel Hiviny , Michelini Lisete Compagno
Published: June 2021

We evaluate the participation of nitric oxide (NO) in the glutamatergic neurotransmission of rostroventrolateral medulla (RVLM) of awake rats sedentary or exercise trained. 33 Wistar rats weighting 220 to 250g were submitted to swimming training (Gtr) or sedentary (Gsed). After the last training session guide cannulae were implanted to the RVLM and after 3 days of recovery, cannulas to the femoral artery and vein were implanted for direct recording of mean arterial pressure (MAP) and heart rate (HR) 24 hours later with animals awake. Results showed bradycardia at rest for the Gtr compared to Gsed (GTr: 326.67±7.3 vs. Gsed: 349.63±4.9bpm) and a greater baroreflex gain (Gtr: −4.41±0.5 vs. Gsed: −2.42±0.31). The pressor response to glutamate in Gsed was increased compared to Gtr (Gsed: ÄMAP: 53±7.7mmHg vs. Gtr: ÄMAP: 32±4.2mmHg) and indifferent after microinjection of carboxy‐PTIO (Gsed: ÄMAP: 41±7.7mmHg vs. Gtr: ÄMAP: 39±5.9mmHg). The pressor response of glutamate in the Gsed before microinjection of n‐propyl was also increased compared to Gtr (Gsed: ÄMAP: 49±3.8mmHg vs. Gtr: ÄMAP: 36±3.9mmHg) and did not change after n‐propyl (Gsed: ÄMAP: 42±5mHg vs. Gtr: ÄMAP: 41±5.2mmHg). The data indicate that swimming can decrease sympathetic activity modifying glutamatergic neurotransmission in the RVLM. The removal of NO reversed those alterations, suggesting a role of NO in the RVLM during exercise training.
Analysis of hepatic transferrin receptor‐1 (TFR1) demonstrates appropriate iron‐sensing in old rats after environmental heat stress
Authors: Bloomer Steven
Published: June 2021

Elevations in hepatic iron content occur with aging and physiological stressors, which may promote oxidative injury to the liver. The cellular mechanism for this increase in iron is unclear, but may involve differential regulation of iron import proteins. Transferrin receptor‐1 (TFR1) facilitates iron influx by binding diferric transferrin, while divalent metal ion transporter‐1 (DMT1) is thought to mediate influx of non‐transferrin‐bound iron. The purpose of this study was to evaluate a role for these iron transport proteins in the transient increase in hepatic iron after heat stress in old rats. Young (6 mo) and old (24 mo) Fischer 344 rats underwent a two‐heat stress protocol and livers were harvested at 0, 2, and 24 h after the second heat stress. Immunoblot analysis was utilized to evaluate the expression TFR1 and DMT1. Under control conditions, expression of both TFR1 and DMT1 was significantly lower in old, compared to young rats. After heat stress, TFR1 declined in the old rats, but was unchanged in the young cohort. Hyperthermia did not affect expression of DMT1 in either age group. Since TFR1 is inversely regulated by intracellular iron, these results demonstrate that after heat stress, old rats mount an appropriate response to an increase in hepatic iron. The results also suggest that TFR1 is sensitive to acute changes in iron, while DMT1 seems responsive only to more chronic changes in hepatic iron content.
Hepcidin and Ferroportin in Different Murine Models of Obesity Challenged with Polymicrobial Sepsis
Authors: Singer Georg , Ruedl Rebecca , Sagmeister Manfred , Deutschmann Andrea , Granger D Neil
Published: June 2021

Sepsis is a leading cause of mortality in non‐coronary intensive care units. Obesity has been on the rise over the past decades. Hepcidin diminishes ferroportin expression and decreases ferritin levels. In both obesity and sepsis hepcidin is increased causing lower serum iron levels. The changes of hepcidin expression in murine obesity and sepsis are unknown. The objective of this study was to compare hepcidin, ferroportin and ferritin levels in murine models of obesity (ob/ob and db/db) challenged with cecal ligation and puncture (CLP) to values in corresponding shams. RT‐PCR and ELISAs were used to quantify the expression of hepcidin and ferroportin in hepatic and subcutaneous fat tissue, and serum levels of hepcidin and ferritin in WT, ob/ob and db/db mice 6 hours following CLP. CLP led to increased hepatic expression of hepcidin in WT and ob/ob, but not db/db, mice. Hepcidin expression in subcutaneous fat was elevated solely in db/db sham mice. Ferroportin expression was unchanged in septic obese mice. Serum levels of hepcidin were significantly decreased in ob/ob and db/db CLP mice and their corresponding shams compared with WT sham and CLP mice. Ferritin levels were unchanged in all groups. The elevated tissue expression of hepcidin following CLP does not appear to exert a systemic impact, as indicated by the unchanged serum levels of hepcidin and ferritin (supported by the FWF Austrian Science Fund, P22567, Government of Styria).
Drosophila as a model for human infection of Shigella flexneri
Authors: Maldonado‐Contreras Ana , Jones Rheinalt M. , Silverman Neal , McCormick Beth A.
Published: June 2021

Shigella flexneri is a Gram‐negative human adapted pathogen that causes bacillary dysentery. A hallmark of Shigella infection is the ability to invade the colonic epithelium, which requires a 220 kB virulence plasmid. Encoded on the plasmid are the components of a type‐III secretion system that contribute to host cell invasion and virulence. While invasion has been thoroughly investigated, understanding of the host immune response to Shigella infection and how Shigella subverts aspects of this response is not well understood due the lack of appropriate animal models. Our objective is to establish Drosophila melanogaster as a suitable model to study the innate immune response against S. flexneri infection. To mimic human infection, we established a feeding method whereby Drosophila ingests S. flexneri orally. Sterile filter disks were soaked in a solution containing sucrose, yeast and fresh Shigella (108–109), and were placed on top of normal fly food. Using this method we determined that ingestion of S. flexneri produces an intestinally localized lethal infection that is dependent on the Shigella virulence plasmid. Moreover, the survival rate of flies exposed to S. flexneri was found to be dose‐dependent. These results indicate that Drosophila is susceptible to S. flexneri infection and can be further exploited as a model to study the key elements involved in the innate immune response against this infection. Funding: NIH