bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of β‐Catenin Signaling by 3‐Acetylaltholactone in Cancer Cells
Authors: Boonmuen Nittaya , Chairoungdua Arthit , Tuchinda Patoomratana , Piyachaturawat Pawinee
Published: November 2023

Wnt/β‐catenin signaling pathway is a complex protein interaction network which plays important functions in cell proliferation, survival and differentiation during animal development and in disease conditions. In Wnt pathway, binding of Wnt ligand to its receptors at the plasma membrane results in inactivation of the β‐catenin destruction complex and promotes the accumulation of β‐catenin translocation from cytoplasm into the nucleus to initiate the transcription of Wnt‐responsive gene expression by binding with TCF/LEF transcription factor. The aberrant regulation of Wnt/β‐catenin signaling has been found to associate with many human diseases including cancers. Therefore, it has been highlighted as a promising target for anti‐cancer drugs. In the present study, we reported the inhibitory effect of 3‐acetylaltholactone, a plant‐derived compound from Goniothalamus sp on the Wnt/β‐catenin signaling pathway in cancer cells. The compound inhibited growth of breast cancers (MCF‐7, and MDA‐MB‐231) and endometrial adenocarcinoma (Ishikawa cell lines). It effectively decreased the expression of β‐catenin protein in breast cancer cells and inhibited TCF/LEF mediated transcriptional activity in HEK293 cells. In addition, 3‐acetylaltholactone also decreased the mRNA expressions of Wnt signaling target genes in MCF‐7 cells. Moreover, it inhibited cell migration by using wound healing assay in MDA‐MB‐231 cells, a highly metastatic human breast carcinoma cell line. In conclusion, these results suggest that 3‐acetylaltholactone mediates anticancer effect on breast cancer cells through inhibition of the Wnt/β‐catenin signaling pathway.

Support or Funding Information

Supported by Mahidol University, TRF through RGJ‐ Ph.D. Program (PHD/0170/2552 to PP and NB), and IRN58W0004.
Ecologically‐relevant Temperatures and Stress in Desert Pupfish
Authors: McKenna Austin , McKenna Ken , Hillyard Stanley , van Breukelen Frank
Published: November 2023

The Southwest United States was much wetter in its recent past. For instance, Death Valley was inundated with the 100m deep Lake Manley just 10,000 years ago. As the waters receded, pupfish were isolated to the warm springs and spring outflows that characterize their current habitats. These fish spent much of their recent evolutionary history in cooler waters and now reside in waters that may be > 33 °C. We asked if rearing or acclimating fish at the ecologically relevant temperature of 33 °C would result in stress as compared to when fish are reared at 28 °C. Heat shock protein 90 was increased 4 fold during both a 4 h acute exposure as well as >3 week chronic exposure to 33 °C. Similarly, hypoxia‐induced factor alpha was increased ~3X at 33 °C. However, cortisol was reduced 2‐fold in 33 °C chronically acclimated fish but not in acute exposure as compared to 28 °C acclimated controls. The Cdk inhibitory protein, p21 was increased during acute exposure but not during chronic exposure to 33 °C. Taken together, these data suggest that stress may be differentially managed and processed in context. For instance, while it may be possible for p21 to repress cell cycle for 4 h, it is less likely that cell cycle may be inhibited for 3 weeks even in the face of environmental insult.
Spinal Oxygen Sensors in Larval Amphibians
Authors: Evanger Iver J , Reed Mitchell D , Wilson Richard JA , Dutschmann Mathias , Taylor Barbara E , Harris Michael B
Published: November 2023

We have recently identified mammalian spinal preganglionic sympathetic neurons as spinal oxygen sensors (SOS). These cells are excited by hypoxia, silenced by hyperoxia, and contribute to oxygen‐sensitive sympathetic responses important for cardiovascular regulation. In general, terrestrial vertebrates experience acute or chronic hypoxia only during pathophysiological states. In contrast, aquatic vertebrates routinely experience environmental hypoxia and hypoxic sensitivity is critical to homeostatic regulation. The aim of the current study is to identify SOS neurons in aquatic larval amphibians. We test the hypothesis that endogenously hypoxia‐sensitive neurons are present in the spinal cords of larval bullfrogs (Lithobates catesbeianus). Spinal cords were isolated from bullfrog tadpoles and incubated for 8 hours in either artificial cerebrospinal fluid or high Mg/low Ca solution to synaptically maintain or isolate cells. Preparations were then exposed to either hyperoxia, normoxia or hypoxia (PO2 of 700, 300 or 100 mmHg respectively) for 3 hours, after which tissues were fixed, sliced and stained to reveal early gene protein expression (c‐Fos) characteristic of cellular activation. Results illustrate the presence of hypoxia‐activated neurons in both synaptically intact and isolated preparations, indicating SOS neurons in amphibian spinal cords. We are characterizing the identities of these cells.

Support or Funding Information

Work reported in this publication was supported by the National Heart Lung and Blood Institute and National Institute of General Medical Sciences of the National Institutes of Health under awards 1R15HL126105, 1SC2GM112570 and three linked awards number RL5GM118990, TL4GM118992 and 1UL1GM118991. Additional support was provided by the Natural Science and Engineering Research Council of Canada, the Canadian Institutes of Health Research and Alberta Innovates ‐ Health Solutions (RJAW) and the Australian Research Council (MD). The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health or any other funding body.
Oxygen binding affinities and effector sensitivities of hemoglobins of African mole rats inhabiting hypoxic and hypercapnic burrows
Authors: Weber Roy E. , Jarvis Jennifer U.M. , Fago Angela , Bennett Nigel C.
Published: November 2023

African mole rats (family Bathyergidae) that excavate and inhabit deep, complex and sealed underground galleries may become subjected to severe hypoxia and hypercapnia (O2 tensions falling below that at the summit of Mount Everest, and CO2 tensions up to 10% of barometric pressure) that predictably impose austere constraints on the exchange and internal transport of respiratory gases. In contrast to high‐altitude mammals, little information is available on possible adaptations in hemoglobin (Hb) function that may mitigate these constraints. Aiming to identify the cellular and molecular mechanisms that safeguard tissue O2 supply in these mole rats, we measured the hematological characteristics as well as Hb‐O2 affinity and it's sensitivity to pH (the Bohr effect), CO2 tension, 2,3‐diphosphoglycerate (DPG, the major organic cofactor modulating Hb‐O2 affinity in mammalian red cells) and to temperature, in six (two strictly‐solitary and four social) species of mole rats originating from a range of Southern African biomes and soil types. The Hbs of the different species showed slight variation in O2 affinity and its sensitivity to pH, DPG and temperature, that did not correlate with soil‐type and sociality, but exhibited conspicuous and variable reductions in specific (pH‐independent) CO2 effects compared to other mammalian Hbs, which may be of adaptive significance in safeguarding pulmonary Hb‐O2 loading in hypercapnic burrows. The observations align with the contention that blood O2‐affinities of burrowing mammals are pre‐adapted to high altitude. Attempts are made to correlate the measured oxygenation traits with available amino acid sequences for mole rat Hbs.
Relative Blood Flow and Cardiac Output in Embryos of the Snapping Turtle Chelydra serpentina (Reptilia; Chelonia)
Authors: Sartori Marina R. , Kohl Zachary F. , Abe Augusto S. , Taylor Edwin W. , Crossley Dane A.
Published: November 2023

In embryos from egg‐laying amniotic vertebrates gas exchange takes place over the chorioallantoic membrane (CAM), an extra‐embryonic circulation analogous to the mammalian placenta, and the oxygen obtained from the environment is convectively transported to the tissues of the developing embryo. Blood perfusion of the CAM (QCAM) and embryonic (QEMB) circulation can be regulated through blood shunts to or from each location. We used colored polystyrene microspheres to measure relative distribution of blood flow between QCAM and QEMB of the snapping turtle, Chelydra serpentina at different points of incubation at 30°C. We examined the embryonic capacity to regulate and redistribute the fractions of cardiac output by α‐blockade and acute 10% hypoxia exposure. Absolute flow was estimated based on measurements of arteriovenous oxygen difference (A‐V diff) according to the Fick Principle. QCAM was 9 (0.33 ml/g/min), 31 (0.17 ml/g/min), and 26% (0.07 ml/g/min) and QEMB was 84 (3.08 ml/g/min), 66 (0.36 ml/g/min), and 72% (0.19 ml/g/min) at 50, 70, and 90% of incubation, respectively. Flow directed to the yolk remained relatively low in all periods. α‐blockade had no influence on blood flow redistribution. Acute hypoxia elicited a bradycardia and a redistribution of the cardiac output fractions among embryonic tissues. QCAM was reduced (from 37% to 23%), and flow increased both to the liver (from 3% to 6%) and to the brain (from 0.8% to 2%) at 70% of incubation. At 90%, flow to the brain doubled during hypoxia. Our results suggest that cardiac output distribution during normoxia is preferentially directed to specific tissue beds, likely dictated by different oxygen demands during tissue differentiation and growth. Embryonic snapping turtles also showed a marked capacity of blood flow regulation during acute hypoxia, primarily protecting flow to the brain, an oxygen sensitive organ.

Support or Funding Information

FAPESP 2014/01666‐5
Arterial PO2 Values Vary Dramatically During Diving and Surface Activities in the Freshwater Turtle (Trachemys scripta)
Authors: Williams Cassondra L , Hicks James W
Published: November 2023

Mammals and birds maintain a narrow range of arterial PO2 values to preserve near complete O2 saturation. Since arterial blood gases in mammals and birds are tightly coupled with lung gas values, this is accomplished through changes in ventilation. In reptiles, pulmonary and system circulations are not completely separated and thus, cardiac shunts play a role in arterial blood gas values. Increases or decreases in the degree of right to left shunting (blood flow bypassing the pulmonary system) will immediately alter admixture and arterial PO2 can change rapidly despite stable ventilation. Further, anoxia‐tolerant reptiles, such as the red‐eared slider, may not need to maintain high arterial PO2 values. As a result, blood oxygen levels may vary more than in mammals and birds during diving as well as during other activities. To investigate this question, we continuously measured arterial PO2 in undisturbed, untethered red‐eared sliders (Trachemys scripta) over a 48‐hour period. Our hypotheses were (1) during routine activities, arterial PO2 values will vary but remain at or above 40 mmHg (75% O2 saturation) and (2) end of dive arterial PO2 values will be lower with longer submergences. Six adult red‐eared sliders (1546 ± 144g) were kept in tanks filled with 25°C water in a 25°C room. PO2 electrodes were surgically implanted in the carotid artery of anesthetized turtles. A self‐contained, waterproof data logger attached to the PO2 electrode was programmed to record PO2 at 1 Hz and epoxied to the carapace. After recovery, turtles were returned to the tank where they could freely swim and bask on a platform warmed by a heat lamp. Turtle behavior was recorded using a surveillance camera placed above the tank. Data were collected and analyzed. All procedures were approved by the University of California (IACUC #2011‐2995). Arterial PO2 during routine activities varied dramatically and individual PO2 values were often less than 40 mmHg. Mean PO2 values for each activity were: 22–42 mmHg for resting underwater; 23–55 mmHg while resting at surface; 29–49 mmHg during basking; and, 28–41 mmHg while swimming. Minimum PO2 values during surface events, basking, and swimming were often as low as minimum vales during submergences. End of submergence PO2 values varied dramatically from 2.7 to 51.7 mmHg and were significantly related to duration in only two turtles (p=0.007, 0.009). These results suggest freely diving, undisturbed T. scripta do not tightly regulate arterial O2 levels or maintain stable high arterial PO2 values even at the surface. The drive to maintain high arterial PO2 levels may be absent in these turtles due to their high tolerance to hypoxia and minimal metabolic demands.

Support or Funding Information

This research was funded by NSF IOS 1121324 (to J. Hicks). C.L.W. was supported in part by a National Institute of Health grant (NIH‐NIAMS T32AR047752 to V. Caiozzo).
Anatomic Intrapulmonary Arteriovenous Anastomoses And Right‐To‐Left Shunt As Evaluated By The Multiple Inert Gas Elimination Technique
Authors: Stickland Michael K , Wagner Peter D , Tedjasaputra Vincent , Fuhr Desi P , Collins Sophie E , Wagner Harrieth , van Diepen Sean , Byers Brad W , Hopkins Susan R
Published: November 2023

Background

Previous work using agitated saline contrast echocardiography suggests anatomic intrapulmonary arteriovenous anastomoses (IPAVAs) can be recruited during exercise, in hypoxia and when cardiac output is increased pharmacologically. These observations are in contrast to the considerable work using the multiple inert gas elimination technique (MIGET) which has not found significant right‐to‐left intrapulmonary shunt at rest or during exercise. The purpose of the present study was to evaluate anatomical intra‐pulmonary arteriovenous connections using agitated saline contrast echocardiography and 25μm microspheres and compare to right to left gas exchange shunt quantified by MIGET in the dog.

Methods

Dogs were anaesthetized (I.V. propofol), ventilated (5 cm H2O PEEP) and evaluated during the following conditions: 1) Baseline, 2) 10 μg/kg/min I.V. dobutamine, 3) 2 μg/kg/min I.V. dopamine, 4) hypoxia (FIO2 = 0.125). Within each condition, pulmonary mixed venous blood was obtained via a Swan‐Ganz catheter, and arterial blood via indwelling femoral catheter. Ventilation‐perfusion mismatch (VA/Q.), diffusion limitation and right‐to‐left shunt was quantified by MIGET and cardiac output by direct Fick. Presence of IPAVAs was evaluated by agitated saline contrast echocardiography, as well as contrast echocardiography using 25μm microspheres.

Results

At baseline, no animal demonstrated a positive contrast echocardiogram suggestive of IPAVAs, and calculated right‐to‐left shunt from MIGET was 0.3±0.1% of cardiac output. Across the three experimental conditions, saline contrast bubbles were detected in the left ventricle 83% of the time, while 25μm microspheres were detected in the left ventricle by echocardiography 67% of the time. No diffusion limitation or low VA/Q. regions (i.e. V.A/Q. ratio of 0.005–0.1) were detected at any point, and right‐to‐left shunt was below 1% during dobutamine, dopamine, and hypoxia (0.6±0.3%, 0.6±0.3%, 0.8±0.6%, respectively).

Conclusion

Anatomic arteriovenous anastomoses of approximately 25 μm in diameter can be recruited during hypoxia and pharmacologically in the healthy intact lung; however, their functional contribution to right‐to‐left shunt or gas exchange impairment is minimal.

Support or Funding Information

Natural Sciences and Engineering Council of Canada (NSERC)
Inhibitory glial modulation of excitatory neurotransmission is reduced in nucleus tractus solitarius (NTS) of rats after short‐term sustained hypoxia
Authors: Accorsi‐Mendonca Daniela , Bonagamba Leni GH , Machado Benedito H.
Published: November 2023

During hypoxia condition the peripheral chemoreflex is activated and induces autonomic and respiratory responses. In this study we analyzed the effect of short‐term sustained hypoxia [(SH) 24 hours, FIO210%] on the synaptic transmission of NTS. Using brainstem slices and whole‐cell patch clamp we observed that passive properties of NTS astrocytes were not affected by SH [input resistance: control: 44.5 ± 14 MΩ (n=8) vs SH: 31.5 ± 10 MΩ (n=4)][RMP control: −84 ± 3.6 mV (n=10) vs SH: −75 ± 5.2 mV (n=7)]. Since protease‐activated receptors 1 (PAR1) are found only in NTS astrocytes and not in neurons, we also evaluated the glial modulation on neurotransmission by activation of astrocytes with SFLLRN‐NH2 (PAR1 agonist, 20 μM). The SFLLRN‐NH2 decreased the amplitude of evoked excitatory post‐synaptic currents (eEPSCs) in NTS neurons from SH animals to values similar to the control group [control: −51 ± 5 pA (n=3) vs SH: −56 ± 8 pA (n=5)]. SFLLRN‐NH2 did not change the RMP of NTS neurons in both groups [control: aCSF: −65.5 ± 1.5 mV vs aCSF + SFLLRN‐NH2: −61 ± 5 mV, (n=4); SH: aCSF: −63 ± 5 vs aCSF + SFLLRN‐NH2: −56 ± 8 mV, (n=4)]. In addition, using in vivo labeling and multi‐photon microscopy, we observed that SH decreased the cellular density of astrocytes in NTS [control: 0.003 ± 0.0004 (n=5) vs SH: 0.001 ± 0.0001 cells / μm2 (n=5)]. In conclusion, SH decreased the number of astrocytes and their inhibitory modulation on excitatory transmission on the NTS neurons.

Support or Funding Information

Financial support: FAPESP and CNPq.
Bicuculline sensitive tonic current in RVLM neurons
Authors: Gao Hong , Derbenev Andrei V
Published: November 2023

Neurons in the rostral ventrolateral medulla (RVLM) are best known for controlling arterial blood pressure and sympathetic outflow. It is well accepted that GABAergic inhibition in the RVLM is critical for the control of sympathetic outflow, and our previous study demonstrated that extrasynaptic GABAA receptors tonically inhibit RVLM neurons. In order to identify the subunit composition of GABAA receptors mediating the tonic current, we conducted whole‐cell patch‐clamp recordings from presympathetic RVLM neurons identified with retrograde viral labeling. First, we determined the effect of zolpidem on the amplitude of tonic current. Application of zolpidem (1μM), which activates GABAA receptors containing α1, α2 and/or α3 subunits, failed to modulate the amplitude of tonic current. Second, to determine the contribution of δ subunits containing GABAA receptors to the tonic current, we used THIP (4,5,6,7‐tetrahydroisoxazolo(5,4‐c)pyridin‐3‐ol), an agonist of δ subunits containing GABAA receptors. Application of THIP (10μM) produced no changes in the amplitude of tonic current in RVLM neurons. Then, a non‐selective voltage‐dependent K+‐channels blocker, 4‐aminopyridine (4‐AP), and a voltage‐dependent transient outward K+‐channels blocker, quinidine, was used. 4‐AP and quinidine induced a significant shift in the baseline current, corresponding to a 32.5±3.3pA and 78.5±8.5pA current, respectively. This data demonstrate that the tonic current sensitive to bicuculline methiodide in the RVLM is partially mediated by time‐ and voltage‐dependent transient outward K+‐channels, which may regulate the initial phase of repolarization modulating the voltage‐ and time‐dependence of action potentials in RVLM neurons.

Support or Funding Information

This work was supported by NIH HL122829 for AVD.
Blockade of orexin receptors reduces glutamatergic excitation of adrenal premotor neurons in the rostral ventrolateral medulla
Authors: Korim Willian Seiji , Verberne Tony J
Published: November 2023

Excitation of adrenal sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM) elicits epinephrine release as part of the glucose counter‐regulatory response to hypoglycemia. It is known that this response is partly mediated by excitatory inputs from hypothalamic orexin neurons. Although some orexin neurons co‐express glutamate, whether orexin and glutamate interact in order to facilitate adrenal sympathetic premotor neurons is unknown. In pentobarbitone‐anesthetized male Sprague‐Dawley rats, we show tested whether glutamatergic activation of adrenal premotor neurons depends on activation of orexin receptors (Ox‐R). Microinjection of glutamate (50 mM/50 nl) into the RVLM, where adrenal premotor neurons are found, increased adrenal sympathetic nerve activity (ASNA). The increase in ASNA was reduced following non‐selective blockade of orexin receptors. RVLM orexin receptors were blocked by microinjecting a combination of SB334867 (Ox1‐R antagonist) and TCS OX2 29 (Ox2‐R antagonist) into the RVLM. The reduction in ASNA in response to glutamate microinjection into the RVLM was dose dependent (0.1–10 mM/50 nl), and the response was abolished with 1 mM of the combined antagonists (P<0.05). The results suggest that the counter‐regulatory release of epinephrine during hypoglycemia depends on the interaction between glutamate and orexin receptors located on the adrenal sympathetic premotor neurons.

Support or Funding Information

NHMRC Australia

Austin Medical Research Foundation
Insulin‐responsive Adrenergic Neurons in Rat Medulla Oblongata
Authors: Llewellyn‐Smith Ida J , Senthilkumaran Manjula , Bobrovskaya Larisa , Verberne Anthony J M
Published: November 2023

Adrenergic neurons in the brainstem play critical roles in glucose homeostasis. When glucose availability is low, these neurons drive secretion of adrenaline from the adrenal medulla and food intake. Although glucoprivation‐sensitive adrenergic neurons in the brainstem have been mapped, the distribution of adrenergic neurons that respond to insulin has not been reported. To fill this gap in knowledge, we fasted rats overnight and then gave them intraperitoneal injections of either insulin (10 IU/kg) or saline after 10 days of twice‐daily handling. Tail vein blood samples were collected immediately before injection and at 30, 60 and 90 min after injection, at which time the rats were perfused with 4% formaldehyde. Coronal sections of perfused medulla (1:4 series) from the spinomedullary junction to mid‐facial nucleus were immunoperoxidase stained to show Fos‐immunoreactivity (IR) with a black reaction product plus phenylethanolamine N‐methyltransferase (PNMT) or choline acetyltransferase (ChAT)‐immunoreactivity with a brown reaction product. Insulin treatment lowered blood glucose from 4.9+0.3 mmol/L at baseline to 1.7+0.2 mmol/L at 90 min (mean + SEM; n=6) whereas saline treatment had no effect (4.9+0.2 mmol/L at baseline vs 5.2+0.4 at 90 min; n=3). In insulin‐treated rats, many PNMT‐IR C1 neurons had intensely‐stained Fos‐IR nuclei, indicating that they had been activated. The proportion of Fos‐IR C1 neurons was higher in the caudal than in the rostral part of the C1 cell column. Insulin treatment also induced Fos‐IR in almost all of the rostral C2 neurons and in many of the midline C3 neurons. The insulin‐responsive C2 neurons may be catecholaminergic dorsal vagal motor neurons since ChAT‐positive neurons with Fos‐positive nuclei occurred in the same locations and numbers. In saline‐treated rats, many fewer C1 neurons were stained for Fos and the staining was much less intense. Fos‐IR occurred in almost no C2 or C3 neurons in control animals. These observations indicate that all three groups of medullary adrenergic neurons are activated by insulin. The high numbers of PNMT‐positive neurons that strongly express Fos after insulin treatment support the important roles that these neurons play in the central response to a decrease in glucose bioavailability.

Support or Funding Information

Diabetes Australia Research Trust; National Health and Medical Research Council of Australia
ENDOCANNABINOID SIGNALING IN THE DORSAL PERIAQUEDUCTAL GRAY INFLUENCES THE DEVELOPMENT OF NEUROPATHIC PAIN
Authors: Dean Caron , Hillard Cecilia , Seagard Jeanne , Hopp Francis , Hogan Quinn
Published: November 2023

Descending endocannabinoid modulation of sensory pathways from the midbrain dorsal periaqueductal gray (dPAG) contributes to the relief of neuropathic pain. Endocannabinoids also activate the sympathetic nervous system through actions at cannabinoid 1 receptors (CB1Rs) in the dPAG. These studies were designed to determine if endocannabinoid signaling in the dPAG influences the development of pain and autonomic changes following nerve injury. Male Sprague‐Dawley rats (320–340 gm) were implanted with telemetric transducers to monitor blood pressure. After one week, either a neuropathic pain model of spared nerve injury (SNI) or sham surgery was performed. Heart rate (HR) and HR variability were derived from blood pressure recordings taken on days 5–7 following implantation surgery and on days 1,3,7,10,14 and 21 following SNI or sham surgery. On these days, rats were also tested for hyperalgesia responses to noxious mechanical stimulation of the hindpaw. Animals that demonstrated a hyperalgesia response rate >20% on day 21 were considered hyperalgesic. Brains were removed on day 21 post SNI/sham, and the dPAG region was excised and quick‐frozen in liquid nitrogen. Real time RT‐PCR was performed to analyze the tissue samples for mRNA expression of the endocannabinoid metabolic enzymes fatty acid amide hydrolase (FAAH) and for CB1Rs. Transcript levels were plotted against blood pressure, HR, autonomic activity, and hyperalgesia response rate for regression analysis to determine correlations. Over 21 days, a range of hyperalgesia responses from 0–80 % developed in SNI rats and no hyperalgesia developed in sham animals. Hyperalgesia animals had a significantly depressed low frequency/high frequency (LF/HF) ratio of HR variability, an indicator of sympatho‐vagal balance and higher HF power, indicative of vagal tone. The hyperalgesia response rate was correlated, and the LF/HF power was inversely correlated, to amounts of FAAH mRNA. Reduced FAAH expression in non‐hyperalgesia animals would be expected to result in increased concentrations of the endocannabinoid, anandamide. Transcript levels for CB1R were unchanged. Animals with hyperalgesia showed a significantly greater decrease in HR over 21 days than those without, while blood pressure remained unchanged. These data suggest that increased endocannabinoid signaling in the dPAG influences autonomic nerve activity and prevents the development of neuropathic pain.

Support or Funding Information

Supported by Veterans Affairs BX001863 (QHH, CD) and the Research and Education Component of the Advancing a Healthier Wisconsin Endowment at MCW (CJH).
Controlled hypotensive Hemorrhage sensitizes angiotensin II‐elicited hypertension
Authors: Xue Baojian , Beltz Terry G. , Guo Fang , Thunhorst Robert L. , Johnson Alan Kim
Published: November 2023

Hemorrhagic shock is associated with increased renin‐angiotensin system (RAS) activity and elevated levels of proinflammatory cytokines (PICs) in the circulation and in central nervous system (CNS) regions controlling arterial pressure and vasopressin release. Using an Induction‐Delay‐Expression (IND‐DEL‐EXP) experimental paradigm, our previous studies demonstrated that activation of the central RAS and inflammation produced by various challenges sensitizes angiotensin (ANG) II‐elicited hypertension. The present study tested the hypothesis that controlled hypotensive hemorrhage also sensitizes the ANG II‐elicited hypertensive response. Male rats were prepared for telemetry recording of blood pressure (BP). During IND, rats were hemorrhaged (5 ml/rat at 1.5 ml/min) via the jugular vein on two occasions separated by 1 day. The blood was collected in heparin and centrifuged, and the cells re‐suspended in saline for re‐infusion. Hemorrhage lowered BP to ~40 mmHg that was maintained for 30 min after which time the rats were reinfused. After a one‐week DEL, the rats were administrated ANG II (120 ng/kg/min, SC) for 2 weeks (EXP). Hemorrhaged rats had a significantly enhanced hypertensive response to the ANG II infusion when compared to control rats (Δ52.3±3.8 mmHg vs. Δ29.8±2.8 mmHg). RT‐PCR analysis of tissue from the lamina terminalis and the hypothalamic paraventricular nucleus indicated that the ANG II treatment upregulated mRNA expression of several components of the RAS and PICs including angiotensinogen, angiotensin type 1 receptor, angiotensin converting enzyme, TNF‐α, interleukin (IL)‐1β and IL‐6 in both control and hemorrhaged rats. Notably, the increases in PIC expression were greater in hemorrhaged rats than that in control rats. The results indicate that controlled hypotensive hemorrhage sensitizes ANG II‐elicited hypertension and that this is associated with upregulation of the central RAS and of brain PICs.

Support or Funding Information

NIH grants HL‐14388, HL‐98207 and MH‐80241
Neuronal expression of prorenin and the (pro)renin receptor in the paraventricular nucleus of the hypothalamus and brainstem in the central nervous system
Authors: Buller Sophie A , Feng Yumei
Published: November 2023

The renin angiotensin system (RAS) is important in the regulation of blood pressure and body fluid homeostasis. Evidence of a local RAS exists within the brain, with all components being synthesized locally, however, active renin is detected only at very minimal levels. We previously reported that the conversion of angiotensinogen to Ang I and thus to Ang II in the central nervous system, is largely mediated by the renin precursor, prorenin, which undergoes non‐proteolytic activation upon binding the (pro)renin receptor (PRR). Despite evidence of a local RAS system within the brain, it remains unclear as to which cell types express prorenin and the PRR, especially in the cardiovascular regulatory regions of the brain. To address this question, we used an antibody that specifically recognizes prorenin, but not active renin, and PRR for immunofluorescence co‐staining with neuronal (NeuN) and astroglial (glial fibrillary acidic protein, GFAP) cell markers. We found that prorenin and PRR immune‐reactivity is mainly co‐localized with NeuN but not GFAP in the brain hypothalamus and brainstem. More interestingly, prorenin and PRR immune‐reactivity is found in approximately 95% of the vasopressin‐positive and tyrosine hydroxylase‐positive neurons. Finally, using cholera toxin B (CTB) retrograde tracing from the RVLM to PVN, and immune‐labeling of CTB for PVN presympathetic neurons, we found that prorenin and PRR are highly expressed in the presympathetic neurons in the PVN. In summary, prorenin and PRR protein are found majorly in neurons, but not astrocytes in key brain regions involved in the regulation of blood pressure and cardiovascular function. In the PVN, prorenin and the PRR are expressed in the vasopressin‐positive, tyrosine hydroxylase‐positive and presympathetic neurons. These data provide anatomical support to a potential physiological importance of prorenin and PRR in the PVN and brainstem.

Support or Funding Information

NIH/NHBL (1R01HL122770) to Y Feng
Adropin Influences the Excitability of Neurons in the Paraventricular Nucleus
Authors: Loewen Spencer P. , Ferguson Alastair V.
Published: November 2023

Adropin, a peptide hormone encoded by the Energy Homeostasis Associated (Enho) gene, has been observed to have metabolic roles in the periphery but unknown actions within the brain. The hypothalamic paraventricular nucleus (PVN) is an important autonomic control center required for regulating energy balance, and is therefore a potential target for centrally acting adropin. Three subpopulations of neurons exist within the PVN, each of which can be classified based on distinct electrophysiological fingerprints. These populations are the magnocellular (MNC) neurosecretory, parvocellular preautonomic (PA), and parvocellular neuroendocrine (NE) neurons. In the present study, we used whole‐cell current‐clamp techniques to examine the effects of adropin on the excitability of each subpopulation of neurons within the PVN of the rat. Bath application of 10 nM adropin to MNC PVN neurons (n=9) elicited responses in 100% of cells, with 78% depolarizing (mean: 5.9 ± 2.5 mV) and 22% hyperpolarizing (mean: −2.6 ± 0.4 mV). Adropin treatment to PA neurons (n=15) caused 53% to depolarize (mean: 5.1 ± 1.6 mV), 13% to hyperpolarize (mean: −10.4 ± 1.8 mV), and the remaining 34% showing no response. Finally, adropin caused responses in 57% of NE neurons (n=14), with 43% depolarizing (mean: 5.7 ± 2.6 mV) and 14% hyperpolarizing (mean: −3.7 ± 0.7 mV). These results suggest that central adropin may exert its physiological effects through actions on neurons in the PVN.

Support or Funding Information

Supported by: the Canadian Institutes of Health Research
The overactive orexin system may play an important role in the poor sleep quality and its associated cardiorespiratory changes in neurogenic hypertension in Spontaneously Hypertensive Rats
Authors: Briggs Logan , Muggler Corey , Li Aihua
Published: November 2023

Non‐dipper essential hypertensive patients suffer from sleep disturbances marked by many microarousal‐associated increases in blood pressure (BP), and the offspring of non‐dipper hypertension parents also showed a greater number of microarousals compared to the offspring of dipper hypertensive and normotensive control parents. Sleep disorders are closely linked to hypertension, and autonomic dysfunction, specifically altered chemoreflex control of sympathetic activity, may play a prominent role in this relationship. It has been reported that spontaneously hypertensive rats (SHRs) have higher sympathetic activity, elevated central and peripheral chemoreflexes, an impaired baroreflex, and poorer sleep quality. Recent studies have linked the orexin system to neurogenic hypertension in SHRs and showed that: 1) blocking orexin receptors with a dual orexin receptor antagonist can significantly lower BP in SHRs with no effect on BP in normotensive rats, and 2) SHRs have more orexin neurons in the lateral hypothalamus than that of normotensive WKY rats. In this study we ask: 1) are there age‐related sleep disturbances and associated cardiorespiratory changes in wakefulness and sleep in SHRs, and 2) will blocking orexin receptors improve the sleep quality and sleep‐associated cardiorespiratory disturbances in SHRs. SHRs at two ages, young (P28–45), and adult (3‐month), were used in this study. All the rats were instrumented with blood pressure telemeter, EEG and EMG electrodes, and 24hr blood pressure, heart rate and sleep‐wake cycle were recorded and analyzed. The results showed: 1) during the light period, SHRs spent 45% & 46% more time in wakefulness, 27% & 23% less time in NREM and 46% & 48% less time in REM sleep at young and adult age respectively compared to age‐matched normotensive WKY controls rats; 2) SHRs, both young and adult, have significantly decreased circadian‐related sleep‐wake and blood pressure difference; 3) SHRs have more sleep disturbance, microarousals and associated increases in BP than normotensive WKY control rats; 4) young SHRs appeared to spend more time in wakefulness than adult SHR during the light period (P=0.009); 5) blocking orexin receptor with a dual orexin receptor antagonist can significantly increase the time spend in sleep in both light and dark periods. In conclusion, SHRs have poor sleep quality from a very young age, and this may contribute to the progressive of hypertension. We suggest that the poor sleep quality in SHRs is associated with the overactive orexin system, and modulation of orexin system may be beneficial for improving sleep quality and treating hypertension in neurogenic hypertension.

Support or Funding Information

Dartmouth Undergraduate Research Office/ Howard Hughes Medical Institute for Undergraduate Research; NIH HL 28066.
Upregulation of Orexin in the Paraventricular Nucleus Contributes to Salt Sensitive Hypertension
Authors: Huber Michael , Zhu Fengli , Li Ningjun , Chen Qing‐Hui , Shan Zhiying
Published: November 2023

Orexin and orexin receptors are extensively expressed in the hypothalamic paraventricular nucleus (PVN) and the hyperactivity of PVN orexin contributes to increased sympathetic nerve activity (SNA) and elevated blood pressure in several animal models. However, the role of PVN orexin in the development of salt sensitive hypertension (SSHTN) is unknown. In this study, we tested the hypothesis that PVN orexin expression is increased in SSHTN and contributes to the development of SSHTN via regulating vasopressin secretion. Eight‐week‐old Dahl salt sensitive (DS) rats were divided into two groups and were fed either a high salt (HS, 8% NaCl) or normal salt (NS, 0.4% NaCl) diet. Six weeks following diet treatment, rats were anesthetized and their mean arterial pressure (MAP) and sympathetic nerve activity (SNA) response to orexin receptor blockade were recorded. In a separate group of rats PVN orexin mRNA levels were measured. The results showed that HS intake induced significant increases in MAP (HS: 146±2 vs. NS: 112±5; n=5; P<0.05) and PVN orexin mRNA levels (HS: 3.8±0.37 vs. NS: 1.0±0.68; n=3; P<0.05). Bilateral PVN microinjection of orexin receptor 1 (OX1R) antagonist SB408124 (30 pmol/50nl) resulted in a greater reduction in arterial blood pressure in DS rats with a HS intake compared to their cohorts with a NS diet (HS: −16±5 vs. NS: −4±4 mmHg; n=5–6; P<0.05). OX1R blockade also increased splanchnic SNA (HS: 48.2±12.6%) vs. NS: 35.82±9%; P=0.2) and renal SNA (HS: 26.7±7.5% vs. NS: 17.4±9.5%; P=0.2) in DS rats with either HS or NS diet. Incubation of orexin A resulted in a dose‐dependent increase in vasopressin mRNA levels (100nM: 2.9‐fold; 1μM; 5.6 fold; 10μM: 18‐fold) in primary cultured neurons from the hypothalamus containing the PVN of neonatal Sprague Dawley rats. These orexin A‐induced increases of vasopressin mRNA were abolished by OX1R blockade. These results coupled with the evidence that plasma vasopressin is upregulated in DS rats under the condition of HS‐induced hypertension suggest that increased PVN orexin expression may be involved in the development of SSHTN through regulating vasopressin secretion.

Support or Funding Information

This study is supported by AHA 11SDG7420029 and NIH R15‐HL122952.
Improvement of Vagal Tone and Reduction in the Blood Pressure by Physical Training in Aging Spontaneously Hypertensive Rats
Authors: Dellacqua Laís Oliveira , Michelini Lisete Compagno , Hindmarch Charlie , Paton Julian Francis Richmond , Murphy David , Antunes Vagner
Published: November 2023

The mechanisms by which physical exercise improve cardiovascular fitness with age are poorly understood. Here, we evaluate the hemodynamic effects of aerobic training and physical inactivity in aging (12‐months‐old) spontaneously hypertensive rats (SHR) compared to normotensive Wistar Kyoto (WKY) rats. Animals from each strain were divided into two groups: trained (T) and sedentary (S). The exercise training lasted 8 weeks (1h/day, 5 days/week), with adjustments of speed to 50–60% of the maximum test. Blood pressure (BP) and heart rate (HR) were recorded from the femoral artery and vein and activation of the baroreflex was performed with bolus injections of phenylephrine and nitroprussiate. BP, HR, spectral analysis and baroreflex analysis were performed at week zero (T0), just before the training protocol, in the second (T2) and eighth (T8) week of exercise training. The performance on the treadmill of trained WKY animals was higher both in the T2 (1.01±0.05 km/h, n=18) and in the T8 (1.04 ± 0,06km/h, n=18) when compared to their respective controls S (0.55 ± 0.05, n=27 and 0.35 ± 0,03km/h, n=27). For SHR the performance on the treadmill of T SHR, were also higher both in the T2 (1.52±0.1km/h, n=18) and in the T8 (1.27 ± 0,04km/h, n=18) when compared with their S controls (1.1 ± 0.14, n=27 and 0.87 ± 0,18km/h, n=27). Systolic blood pressure (SBP) of T8 SHR was lower (155±10 mmHg, n=5) than in control group S8 (198±10 mmHg, n=6). The baroreflex sensitivity in T8 SHR showed a trend of an improvement with no statistical difference when compared to the S8 group. The HF component of the pulse interval was higher in T8 SHR (22.4±5.9 A.U. n=5) than in the control S8 group (6.3±2.4 A.U. n=5). Collectively, our data show that exercise was able to reduce systolic blood pressure, without significantly changing in baroreflex function, and improve the control of cardiac function in the 8 week trained.

Support or Funding Information

Financial Support: FAPESP (# 2012/22368‐7, # 2011/51410‐9).
Pontine and Medullary Sources of Cardiac Vagal Tone
Authors: Farmer David George Stephen , Dutschmann Mathias , Paton Julian F , Pickering Anthony E , McAllen Robin Michael
Published: November 2023

Cardiac vagal tone is a major determinant of health. Here, we investigate the origins of cardiac vagal tone in the working heart‐brainstem preparation (WHBP). The WHBP shows ongoing activity in the cardiac vagal branch (CVB) and a normal pattern of respiratory sinus arrhythmia (RSA). Ongoing activity in the CVB peaks in late inspiration/postinspiration, correlating with the nadir of heart rate. Bilateral inhibition of the pontine Kölliker‐Fuse nucleus (KF) with isoguvacine (GABAA receptor agonist; 10 mM, 50–70nl) abolished RSA at the same time as removing postinspiratory phrenic activity. Mean heart rate rose modestly (baseline: 245.0 ± 9.2 bpm; KF isoguvacine: 270.6 ± 10.0 bpm; p=0.0370; n=8). Cardiac vagal nerve activity continued, however, adopting a tonic discharge pattern with minor early‐inspiratory dips. In sympathetically disabled rats, transection of the brainstem at caudal pontine level had an effect on heart rate that varied between animals (bradycardia or tachycardia) (baseline: 205.6 ± 10.2, pontine transection: 233.9 ± 8.2, p = 0.1024; n=6). Subsequent bilateral inhibition of the nucleus tractus solitarii (NTS) in these animals produced tachycardia (261.5 ± 8.0 bpm, p=0.0020 vs. pontine transection). Atropine (0.1μg/ml) after either KF inhibition or pontine transection followed by NTS inhibition produced tachycardia, confirming that substantial cardiac vagal tone survived those procedures. We conclude that (a) postinspiration is integral to shaping of CVB discharge and to the generation of RSA; (b) a large component of cardiac vagal tone does not require connections to the pons and is independent of central respiratory drive; (c) part of that medullary component depends on neurons in the NTS.

Support or Funding Information

Supported by ARC
Dynamic Gene Regulation Across Physiological Systems Coordinates the Pathogenesis of Hypertension
Authors: Anderson Warren , DeCicco Danielle , Brureau Anthony , Schwaber James , Vadigepalli Rajanikanth
Published: November 2023

The characteristics of hypertension pathophysiology have been extensively studied at levels of analysis ranging from organ physiology to cellular function and molecular expression. However, the dynamics of functional disregulation underlying the pathogenesis of hypertension, both within and across organs, have not been elucidated. We addressed this issue by profiling molecular expression across organs in hypertensive (spontaneously hypertensive rat, SHR) and non‐hypertensive (Wistar Kyoto, WKY) rat strains. We sampled tissue from the brainstem, kidney, adrenal gland, left ventricle, and spleen at ages 4, 6, 8, 12, and 16 weeks. We analyzed expression of 22 genes from three functional classes: inflammation (e.g., cytokines and chemokines), renin‐angiotensin signaling (e.g., angiotensin metabolism, angiotensin receptor), and autonomic regulation (e.g., catecholamine metabolism, adrenergic receptors). Analysis of variance revealed a significant three‐way interaction involving rat strain, organ, and age for Il1b (p‐adjusted = 2.2 × 10−6). Post‐hoc analysis revealed a number of significant differences, including downregulation of Il1b in the SHR kidney compared to that of WKY at 16 wk (p = 0.002). To assess whether there were strain‐dependent differences in the temporal profiles of gene expression in individual organs, we performed a time‐series analysis using the Optimal Discovery Procedure (Storey et al., PNAS, 2005). Our analyses revealed that all genes assayed in the brainstem showed differential dynamics in SHR compared to WKY, and the following genes showed strain‐dependent temporal profiles in the kidney: Il1b, Ccl5, Agtr1a, Ren, Ace, Adrb1, and Adra1a (p‐adjusted < 0.05). Twenty genes showed strain‐dependent temporal profiles in the adrenal gland (p‐adjusted < 0.05). To enhance our understanding of the regulatory mechanisms underlying hypertension development, we integrated an empirical dynamic modeling approach with our data analysis. We estimated kinetic parameters governing gene‐gene interactions within and across organs, and we simulated the corresponding models described by ordinary differential equations. Model simulations supported our time‐series data analysis by showing transient expression variations in the 4–8 wk range for many genes in SHR compared to relatively flat temporal profiles for WKY. This analysis revealed a coordinated wave of expression variations both within and across organs in SHR. Our experimental and modeling results elaborate a multi‐organ coordinated gene expression program underlying the pathogenesis of hypertension.

Support or Funding Information

Funding: NIH/NHLBI R01 HL111621
Microglia modulate Ang II induced neuronal activity and neuroinflammation
Authors: Santisteban Monica M , Zubcevic Jasenka , Febo Marcelo , Raizada Mohan K
Published: November 2023

Objective

Our studies have previously established that neurogenic hypertension is associated with microglial activation in cardioregulatory brain centers, such as the paraventricular nucleus of the hypothalamus (PVN). Treatment with minocycline, an inhibitor of microglial activation, attenuates the persistent increase in blood pressure, peripheral and central inflammation, as well as autonomic dysfunction in vivo. In the present study, we tested the hypothesis that microglia play an important role in modulating Ang II induced neuronal activity and neuroinflammation.

Methods

Adult Sprague Dawley (SD) rats were infused with SQ saline or Ang II (200ng/kg/min) and treated with oral vehicle or minocycline (50mg/kg). Manganese‐enhanced magnetic resonance imaging (MEMRI) was performed at 4 weeks of infusion to indirectly visualize neuronal activity across cardioregulatory brain networks. After imaging, brains were isolated for microglia Iba1 immunostaining to establish potential mechanisms underlying altered brain activity. Primary neuronal and mixed neuroglia cultures were prepared from SD pups on day E20–21. Cultures were treated with 20μM minocycline for 1 hour followed by 1μM Ang II for 6 hours on day 5 for mixed cultures or day 10 for primary neuronal cultures.

Results

Under control conditions, MEMRI detected basal activation of the PVN. In rats infused with Ang II, there was a marked increase in manganese accumulation in the PVN, which is likely indicative of increased PVN neural activity; this effect was markedly reduced by the minocycline treatment, an effect associated with decreased microglial activation in the PVN. In vitro, Ang II induced a 40% upregulation of c‐fos in mixed culture (p<0.01), indicative of neural activation, which was effectively blocked by minocycline pretreatment. Additionally, Ang II increased pro‐inflammatory genes mRNA in vitro, including Il1b (1.5‐fold, p<0.001), Tnf (1.4‐fold, p<0.05), Ccl2 (1.2‐fold, p<0.01), and Nos2 (1.8‐fold, p<0.01), indicative of M1 microglial polarization. Minocycline pretreatment was able to reverse these effects in mixed neuroglia cultures. However, in primary neuronal cultures, minocycline only decreased Nos2 (p<0.05), and did not affect c‐fos or pro‐inflammatory gene expression.

Conclusion

These data indicate microglia activation plays an important role in modulating Ang II induced neuronal activity and neuroinflammation in PVN. Furthermore, minocycline indirectly modulates neuronal activity in vivo and in vitro by inhibiting microglial activation.

Support or Funding Information

HL 33610 (MKR)