bluuebunny/crossref_metadata_2025_split huggingface dataset:
K+ Channel Activation Inhibits Allergen-Stimulated ATP Release by Human Bronchial Epithelial Cells
Authors: Srisomboon Yotesawee , Kita Hirohito , O'Grady Scott 
Published: May 2024

Allergens derived from the fungus Alternaria alternata stimulate the release of multiple alarmins including ATP, IL-33 and DNA fragments from human bronchial epithelial cells (hBECs). Oxidative stress induced by Alternaria extract activates ATP release by opening voltage-dependent anion channels (VDAC-1) in the apical membrane and by stimulating exocytosis of ATP containing membrane vesicles. The present study shows that pretreatment of hBECs with K2P channel inhibitors prior to Alternaria exposure significantly increases the initial rate of ATP release and inhibition of ATP loading into membrane vesicles with bafilomycin A1 blocks the response. Furthermore, pretreatment with the K+ channel openers, EBIO (KCa3.1 channels), NS11021 (KCa1.1 channels) and pinacidil (KATP channels) each inhibited the bafilomycin A1-dependent component of ATP release, reducing the initial rate of ATP secretion by ~50%. K2P channel blockers caused depolarization whereas K+ channel openers produced hyperpolarization of the cell membrane. Additionally, K2P channel inhibitors also increased Alternaria-stimulated Ca2+ uptake into hBECs while K+ channel openers reduced the Alternaria response by ~50%. Imaging experiments involving visualization of fluorescently labeled MARCKS proteins in the apical membrane showed that treatment with Alternaria caused a significant decrease in fluorescence intensity, however pretreatment with K+ channel openers, but not K2P channel blockers, inhibited the effect. These results are consistent with recent findings showing that MARCKS proteins bind to the plasma membrane through an electrostatic association with PIP2 and that phosphorylation by PKC or membrane hyperpolarization following K+ channel activation disrupts this interaction resulting in MARCKS detachment from the membrane. MARCKS detachment uncouples the membrane from the actin cytoskeleton, facilitating vesicle docking, fusion and release of ATP. This study was supported by NIH grants (AI128729 and AI169530) to HK and SMO and by USDA-AES 0016134 to SMO. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Gender Specific Effects of Nr4a1 in Obese Mice
Authors: Herring Jacob , Tessem Jeffery 
Published: May 2024

A central aspect of type 2 diabetes disease progression is impaired functional β-cell mass. The hyperglycemic and hyperlipidemic environment present in type 2 diabetes corresponds with impaired beta cell function. The orphan nuclear receptor Nr4a1 is critical for fuel utilization in various tissues, however little is known regarding its function in the β-cell. Nr4a1 expression is decreased in the β-cell of rodent models of type 2 diabetes, as well as in primary human islets from type 2 diabetics. Here we demonstrate sex specific effects of β-cell specific Nr4a1 deletion under high fat chow feeding. While female and male βNr4a1−/− mice were no different than wild type controls when fed a standard chow diet, a clear sex dependent difference is observed when fed a high fat diet. While high fat fed male βNr4a1−/− have improved fasting and non-fasting blood glucose levels and no change in glucose tolerance, high fat fed female βNr4a1−/− mice have impaired glucose tolerance as early as one month after beginning high fat feeding. We also found that βNr4a1−/− female mice have decreased β-cell mass and decreased expression in known targets of Nr4a1 regulation including Eno1 and SDHβ. Our data suggest that Nr4a1 is critical for maintenance of beta cell function, and that loss of Nr4a1 under conditions of obesity and overnutrition in females results in impaired beta cell compensation and function. NIH R15DK124835-01A1. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Phrenic Long-term Facilitation Following Severe Acute Intermittent Hypoxia Is Preserved in Geriatric Male Rats
Authors: A. Burrowes Kayla , B. Marciante Alexandria , R. Perim Raphael , El-Chami Mohamed , S. Mitchell Gordon 
Published: May 2024

Acute intermittent hypoxia (AIH) elicits a form of respiratory motor plasticity known as phrenic long-term facilitation (pLTF). Both moderate and severe AIH elicit phenotypically similar pLTF but do so via completely distinct cellular mechanisms. With moderate AIH (mAIH), pLTF arises from a serotonin-dominant, adenosine-constrained mechanism. In aged rats, mAIH-induced pLTF is impaired due to increased basal spinal adenosine levels, which constrain serotonin-dependent pLTF. With severe AIH (sAIH), pLTF arises from an adenosine-dominant, serotonin-constrained mechanism. Since spinal adenosine levels are elevated in aged male rats, we hypothesized that age would actually enhance sAIH-induced- pLTF. Young (~3.5 month) and aged (~20 month) male Sprague Dawley rats were urethane anesthetized, artificially ventilated, vagotomized, paralyzed, and exposed to sAIH (3, 5-minute episodes; arterial Po2 = 25-30 mmHg). Integrated phrenic nerve burst activity was measured before (baseline), during hypoxic episodes and 60 minutes after sAIH. Neither baseline phrenic burst amplitude, the short-term hypoxic phrenic response nor pLTF magnitude (assessed as % change in phrenic burst amplitude from baseline to 60 minutes post-sAIH) were different in aged versus young rats (120 ± 22% vs. 98 ± 17%, respectively). Thus, aged male rats preserve the capacity for adenosine driven phrenic motor plasticity. Although we cannot conclude that sAIH-induced pLTF is actually increased, age effects on adenosine versus serotonin-driven plasticity are clearly different. NIH HL148030. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Potassium Ion Channel Modulation as a Potential Remedy for Cadmium-induced Hypertension
Authors: McCalla Garsha , Brown Paul , Nwokocha Chukwuemeka 
Published: May 2024

Objective: Hypertension remains a chief non-communicable lifestyle disease that is negatively associated with dietary cadmium (Cd). The exact mechanism of Cd toxicity and the mitigating role of potassium supplementation are not fully known, hence, this study aimed to investigate their effects on the aorta. Hypothesis: We hypothesized that potassium supplementation can modulate Cd-induced vascular dysfunction. Method: Six cohorts of control, Cd-exposed, and potassium supplemented male Sprague-Dawley rats were selected. Hypertension was induced using cadmium chloride (2.5 or 5 mg/kg b.w.). Blood pressure was measured twice weekly. Reactivity to pharmacological agents with and without potassium ion channel modulation was assessed in thoracic aortic rings after eight weeks, using an organ bath set-up. Data: Statistical analysis was done using one-way analysis of variance and the results were reported as mean ± standard error of the mean. The Games-Howell or Bonferroni post hoc test was used for multiple comparisons. Summary of Results: Cadmium significantly (p<0.05) elevated systolic, diastolic, mean arterial, and pulse pressures, reduced (p<0.001) phenylephrine-induced contraction mostly via BKCa channels, and augmented (p<0.05) sodium nitroprusside-induced relaxation via K-ATP, KV, and KIR channels, without affecting acetylcholine-induced relaxation. Potassium supplementation ameliorated these effects via KIR and BKCa channels. Conclusions: The results indicate that modulation of potassium ion channels in the vasculature might provide therapeutic management for environmentally-induced hypertension and should be further explored. Funding source: The Mona Campus Committee for Research and Publications and Graduate Awards, School for Graduate Studies and Research, University of the West Indies. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Genetically-targeted Recordings from Respiratory Chemoreceptor Neurons in the Retrotrapezoid Nucleus of Freely Behaving Mice by Fiber Photometry
Authors: Souza George , Stornetta Daniel , Abbott Stephen 
Published: May 2024

Central respiratory chemoreceptors regulate breathing homeostasis in relation to arterial pH/PCO2. Neurons located in the retrotrapezoid nucleus (RTN) are considered to function as central respiratory chemoreceptors and/or to be part of the neural network that drives the central chemoreflex. RTN neurons are sensitive to changes in pH in vitro under synaptic blockade and in anesthetized preparations with artificial ventilation. However, there is limited information about their activity in vivo in non-anesthetized conditions. Recent studies have shown that Nmb-expressing neurons in the RTN have acid-sensitive properties and are necessary for the respiratory response to CO2. In this study, we hypothesized that RTN- Nmb neurons are activated during hypercapnia in freely behaving condition. To test this hypothesis, we measured the dynamic activity of RTN neurons using a genetically encoded calcium indicator (GCaMP7s) via fiber photometry in non-anesthetized mice. Expression of GCaMP7s was genetically targeted to RTN neurons by microinjection of a Cre-dependent viral vector (AAV1-Syn-Flex-jGCAMP7s) in transgenic Nmb-Cre mice. One month later, the animals were placed in a plethysmography chamber to simultaneously record breathing and GCaMP7s fluorescence from RTN- Nmb neurons in freely behaving conditions. During hypercapnia, RTN neurons were markedly activated, and the effect size was graded to FICO2 (DF/F0 from baseline: 1.9 ± 1.0 % at 0.03, 8.0 ± 4.0% at 0.06 and 12.0 ± 5.0% at 0.09, n=6), and significantly different for each level of CO2 (RM One-way ANOVA, p=0.0012). Importantly, the increase in RTN neuron activity correlates with changes in the FICO2 (p<0.0001, R2=0.97) and respiratory frequency (p<0.0001, R2=0.85). This study shows that RTN neurons are potently activated by hypercapnia in freely behaving conditions, and the response of RTN neurons to CO2 aligns with changes in breathing. These results are consistent with the concept that RTN neurons monitor arterial PCO2/pH and are essential for the hypercapnic ventilatory response. This study was supported by National Institutes of Health (HL148004 ) to Stephen BG Abbott. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Identification and Characterization of Potential Anti-Hypertensive Therapeutic Candidates in Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin 3 (CUL3) Axis
Authors: Kumar Gaurav , Vazirabad Ibrahim , Golosova Daria , Chaihongsa Nisita , Muskus Patricia , Wackman Kelsey , Lu Ko-Ting 
Published: May 2024

Blood pressure (BP) is regulated by a variety of cellular mechanisms. We previously reported the regulation of endothelial nitric oxide (NO)-dependent vessel tone and BP through RhoBTB1-CUL3 mediated proteasomal turnover of Phosphodiesterase 5 (PDE5). We determined that the C-terminal half of RhoBTB1 consisting of two BTB-domains (B1 & B2) and the C-terminal (C) domain of RhoBTB1 can deliver PDE5 to proteasomal pathway. The cellular expression of RhoBTB1 and CUL3 is ubiquitous whereas expression of PDE5 is cell specific. Moreover, vessel tone is regulated through NO-independent mechanisms. Therefore, we hypothesized that RhoBTB1 may have other interacting partners, potentially functioning as regulators of vessel tone and BP in vascular smooth muscle (VSM) cells, through a similar mode of binding as PDE5. Therefore, we utilized ascorbate peroxidase (APEX2) proximity labelling system comprising APEX2 tagged B1B2C domain (B1B2C-APEX2) to identify unknown potential regulators of vasomotor tone in RhoBTB1-CUL3 axis in A7R5 VSM cells. The background of the proteomic screen was minimized by utilizing APEX2-B1B2 which lacks the final C-terminal region of the protein and cannot bind PDE5. We validated the interaction of several identified proteins with RhoBTB1 and the B1B2C domains using co-immunoprecipitation (Co-IP). Our Co-IP data corroborated the proteomic screen as several proteins demonstrated differential interaction with B1B2C and B1B2. Among them, we examined RbFox2, a regulator of mRNA splicing for further investigation. Further data established that RbFox2 is upregulated under the following conditions: 1) pharmacological inhibition of the proteasomal pathway, 2) in CRISPR edited CUL3KO HEK293 cells, 3) in aorta of VSM cell-specific CUL3 deleted mice, 4) by siRNA mediated knock-down of RhoBTB1 in SMCs, and 5) in Ang-II treated SMCs and aorta of Ang-II treated animals. We hypothesized that RbFox2 could modulate actin and actinin-dependent cytoskeletal dynamics, an important regulator of arterial stiffness. First, Co-IP data suggest an interaction between RbFox2 and SMC-specific alpha actinin. Next, siRNA-mediated knock down of RbFox2 altered the actin and actinin-dependent cytoskeletal dynamics in SMCs. Collectively, this establishes the potential regulation of RbFox2 through the RhoBTB1-CUL3 proteasomal pathway and that RbFox2 may play a mechanistic role in arterial stiffness in hypertension. Funding: NIH R35 HL144807 (to Dr. Curt D. Sigmund). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
pHire: A Novel Red Intracellular pH Sensor
Authors: Desrochers Kate , Holmberg Shannon , Holmes Heather , Zhang Lili , Foresto Neto Orestes , Reynolds Carmen , Romero Michael 
Published: May 2024

Maintaining pH is critical for proper cellular and organ function. To measure intracellular pH, we made a novel genetically encoded protein, pHire, that increases red fluorescence with increasing pH. Thus, pHire allows for the coupling of multiple fluorescent tags without spectral overlap. We have expressed pHire in mammalian epithelial cells, Drosophila, and mice. In this study, our goal was to characterize pHire’s spectral properties by expression in human cells (HEK293) and in E.coli. To biophysically characterize pHire, we made a maltose binding protein (MBP)-pHire fusion protein to purify from E.coli. Pelleting individual colony growths revealed pink bacterial pellets which fluoresced with mCherry filters. Rather than purifying the MBP-pHire protein, equal volumes of bacteria were permeabilized with nigericin and high K+ solution for pH 5 to 9. These aliquots were then used to gather the fluorescence excitation and emission spectra of pHire, allowing us to determine the excitation and emission peaks of pHire as a function of pH. Our results showed peaks at Ex579 nm and Em611 nm, or roughly mCherry. Pelleted bacteria could also be fixed in perfusion chambers, revealing the same calibration curve as in mammalian cells and flies. Next, stable HEK293-pHire cells were selected to assay acid-base transport using NH4Cl or CO2/HCO3− in perfusion experiments. These HEK-pHire cells have less fluorescence than MBP-pHire bacteria but show the same Ex/Em peaks. However, an in-frame ATG 5’ to pHire in a mouse (adding 23 amino acids), removed pHire pH-dependence and decreased bacterial fluorescent output. Together these results indicate that pHire is biophysically similar in cells tested but that unstructured N-terminal additions block pH-responses. Funding: R25-DK101405, R21-DK129897. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Alleviating renal injury and mitochondrial dysfunction following hemorrhagic shock
Authors: Kennard Cianna , Calderon Alfredo , Klemcke Harold , Mdaki Kennedy , Marshall Stephanie , Meledeo Michael , Xiang Lusha 
Published: May 2024

Resuscitation with polydatin (PD) has been shown to alleviate mitochondrial damage and ischemia reperfusion injury following hemorrhagic shock. However, whether PD has the same beneficial effect under prolonged hemorrhagic shock with delayed or limited resuscitation is unclear. Using a previously developed rat model of acute kidney injury (AKI) induced by prolonged hemorrhagic shock, we tested the hypothesis that PD preserves renal mitochondrial function, delays the onset of AKI, and decreases mortality following prolonged hemorrhagic shock without full resuscitation. Additionally, we determined whether polyethylene glycol-20k (PEG) can enhance the beneficial effects of PD via improved capillary perfusion and PD delivery into the ischemic tissues. Rats were randomized into 4 groups (n=8/group) based on treatment: saline (vehicle), PD, PEG, and PD+PEG. Trauma was induced in Inactin anesthetized rats with muscle injury and fibula fracture, followed by pressure-controlled hemorrhagic shock (mean arterial pressure (MAP) = 55 mmHg) for 45 minutes. Animals then received an intravenous injection of saline (vehicle), PD, PEG, or PD+PEG. MAP, blood gases, hematocrit, renal blood flow (RBF), and oxygen delivery were monitored for another 2 hours. Glomerular filtration rate (GFR), urinary sodium uptake, renal mitochondrial function, and mortality were recorded at the end of the experiment. The saline group exhibited AKI, renal mitochondrial dysfunction, and 100% mortality. PD alone showed no effect on mitochondrial function, renal injury, or mortality (100%). PD+PEG restored both complex II and IV activities, while PEG improved mitochondrial complex II function but showed no effect on complex IV. Nevertheless, PEG and PD+PEG similarly improved MAP, renal blood flow, renal oxygen delivery, GFR, sodium uptake, and mortality (PEG: 12.5%; PD+PEG: 0%). Our results suggest that polydatin requires suffcient tissue perfusion to exert its beneficial effects on the mitochondrial function of hemorrhagic shock animals. We used clinically meaningful endpoints to demonstrate the utility of PEG as a therapeutic tool for optimizing drug delivery to treat hemorrhagic shock. DoD disclaimer: The views expressed in this article are those of the author(s) and do not reflect the offcial policy or position of the U.S. Army Medical Department, Department of the Army, DoD, or the U.S. Government. Funding: This study was funded by US Army Medical Research and Development Command. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
The Influence of Early Menopause on Baroreflex Sensitivity in Healthy Females
Authors: Anselmo Miguel F. , Nathe Stephany C. , Stokes William , Koebernick Ella , Tahsin Chowdhury Tasnova , Lee Emma J. , Teshome Soliana 
Published: May 2024

Introduction: The risks of hypertension (HTN) and cardiovascular disease (CVD) rise dramatically in females around the typical age of menopause (TM; menopause age ≥46yr). Early menopause (EM; menopause age <46yr) may substantially increase this risk. Attenuated baroreflex sensitivity (BRS) can be indicative of autonomic dysfunction and has been shown to be a marker of CVD risk. Thus, we aimed to investigate how EM would influence BRS. We hypothesized that females who experienced EM would exhibit attenuated BRS compared to females who experienced TM. Methods: 32 females completed two visits: Visit 1: informed consent, medical and health questionnaires to confirm eligibility, including the Minnesota Leisure Time Physical Activity Questionnaire to assess activity levels. Visit 2: Participants rested in a supine position for 10 min. Non-invasive beat-to-beat blood pressure (BP), heart rate (HR), and muscle sympathetic nerve activity (MSNA) were recorded. Blood was drawn to measure sex steroid levels. Cardiovagal (cBRS) and sympathetic (sBRS) BRS were calculated using the sequence method and Ensemble software, respectively. Both parametric and non-parametric methods were used for the statistical analysis. Results: Results are presented as mean ± SD. Participants in the TM (n=16; age: 63±3yrs, body-mass index (BMI): 25±4kg/m2) and EM (n=16; age: 62±4yrs, BMI: 26±4kg/m2) groups were matched for age and BMI. TM and EM had similar levels of estradiol (TM: 3.9±2; EM: 4.4±4pg/mL) and activity levels (TM: 299±177; EM: 208±147kcal/day), but significantly different menopause ages (TM: 51±3; EM: 43±2yrs; p<0.001). TM and EM had similar resting systolic BP (TM: 129±13; EM: 128±20mmHg), diastolic BP (TM: 79±10; EM: 82±14mmHg), HR (TM: 59±7bpm; EM: 62±9bpm), and MSNA (TM: 51±16; EM: 45±11bursts/100heartbeats), p>0.05 for all. Further, cBRS and sBRS were similar between TM (cBRSup: 9.7±3; cBRSdown: 12.3±7; cBRStotal: 12.3±7ms/mmHg; sBRS: -2.7±1bursts/100Hb/mmHg) and EM (cBRSup: 8.7±4; cBRSdown: 11±5; cBRStotal: 10.4±4ms/mmHg; sBRS: -2±1bursts/100Hb/mmHg; p>0.05 for all). Conclusion: Contrary to our hypothesis, these findings suggest that EM may not influence BRS. These findings indicate that completing menopause early may not disrupt autonomic baroreflex function, suggesting that other mechanisms need to be explored to better understand the increased CVD risk in postmenopausal females who complete menopause prematurely or early. This study was supported by a NIH 1 K01 AG064038-01A1 (MLKR), UMN Grant-in-Aid (MLKR), F32HL160012 (EL), and NIH National Center for Advancing Translational Sciences grant UL1TR002494. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.