bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of Estradiol in the Behavioral and Cardiovascular Consequences of Social Stress in Females
Authors: Padi Akhila R , Finnell Julie E , Lombard Calliandra M , Wilson Marlene A , Wilson L Britt , Wood Susan K 
Published: November 2023

Repeated exposure to social stress results in the emergence of psychosocial disorders, which significantly increases the risk of cardiac morbidity. Depressed women are at a greater risk for the development of comorbid cardiovascular disease in comparison to depressed men. Furthermore, increased risk in females begins during puberty and ends following menopause, suggesting that ovarian hormones play a role. In this study, female rats were subjected to witnessing a social defeat encounter between an aggressive male resident and a male intruder. Female rats were implanted with a cardiovascular telemetry device and either ovariectomized (OVX) or sham OVX (intact). In study 1, stress‐induced cardiovascular consequences were measured in female intact vs OVX witness rats compared with controls. In study 2, all females were OVX and replaced with estradiol (E) or vehicle (V). Each witness was paired with an intruder, and placed behind a plexi‐glass partition in the resident cage for the 5 daily 15 min intruder defeat exposures. Cardiovascular telemetry from Study 1 indicated higher heart rate, blood pressure, and the occurrence of premature ventricular contractions (PVCs) and behavioral anhedonia during stress in intact female witnesses compared to OVX witness and controls. This suggests that ovarian hormones increase sensitivity to stress‐induced cardiac dysfunction and stress‐induced depressive‐like behavior. Study 2 identified a higher presence of the same cardiac dysfunction factors during stress in OVX+E witnesses versus OVX+V witnesses. This demonstrates that E plays a role in the increased vulnerability to stress‐induced cardiac and behavioral dysfunction. Ongoing studies are assessing the mechanism whereby, when combined with stress, E increases susceptibility to cardiac dysfunction through analyses of heart rate variability. These findings recapitulate the increased vulnerability to cardiovascular dysfunction in an intact, cycling female population that suffers from comorbid depressive disorders. These studies seek to identify why females who undergo stress are more vulnerable to the development of co‐morbid depression and cardiovascular disease, thereby allowing for the development of novel targeted therapeutics.Support or Funding InformationASPIRE18080‐16‐41861, 15SDG22430017, and P20GM103641
The Timing of Weaning in Rats Has a Long‐Lasting Impact on the Vulnerability to Stress‐Induced Gastric Erosion in Both Sexes
Authors: Filaretova Ludmila , Vataeva Ludmila , Zelena Dora 
Published: November 2023

Weaning is actually a maternal separation, characterized by maternal reduction of resources, therefore is both psychologically and energetically stressful to the offspring. Weaning is an important period of life and its timing may influence the resilence for later stress. One of the most important stress‐related disorder is gastric ulceration. Weaning is actually a maternal separation, characterized by maternal reduction of resources, therefore is both psychologically and energetically stressful to the offspring. Therefore we aimed to investigate the sensitivity of gastric mucosa to cold (at 14°C) water immersion stress (WIS for 3h) in adult (75‐day‐old) female and male rats after weaning them at different timepoints (at 17, 21, 30, 36 or 42 postnatal days). The connection with stress was studied by comparing control groups to those underwent WIS at the time of weaning and measuring corticosterone levels at the time of collecting the stomach samples. The timing of weaning has strong impact on all studied parameters. Stress‐induced erosion development was the smallest in rats weaned at 36‐day independently from preconditioning with WIS at weaning, or sex, despite a clear sex‐effect on blood corticosterone levels and body weight. WIS at weaning influenced only the body weight in adult rats weaned at 30‐day, being higher in stressed than in control groups. Erosion area significantly correlated with blood corticosterone level at weaning non‐stressed females. Taken together our results confirm that the timing of weaning has long‐lasting impact on the resiliance of gastric mucosa to ulcerogenic stressful events. The postnatal day 30–36 seems to be the optimal for weaning as both earlier and later weaning increased vulnerability. In conclusion, in contrast to the general practice, weaning at the postnatal day 30–36 would be optimal for rats leading to maximal mucosal resistance. Additional stress at weaning does not make too much difference.Support or Funding InformationThe study was supported by grant of Russian Science Foundation (RSF) №14‐15‐00790. (Filaretova L.: who was responsible for general experimental design, performing experiments in adult rats and analysis of the results).
Altered Kisspeptin Responsiveness in Bmal1 Knockout Females
Authors: Tonsfeldt Karen J , Schoeller Erica , Brusman Liza , Mellon Pamela 
Published: November 2023

The use of the hypothalamic peptide kisspeptin is a novel treatment for hypothalamic infertility. The preovulatory luteinizing hormone (LH) surge occurs in the hypothalamic‐pituitary‐gonadal axis to initiate ovulation. Estrogen primes kisspeptin (Kiss1) neurons in the anteroventral periventricular nucleus (AVPV) to secrete kisspeptin, which potently activates gonadotropin‐releasing hormone (GnRH) neurons to release a pulse of GnRH, eliciting a surge of LH to prompt ovulation. A critical component of this preovulatory surge is temporal gating. Knockout of Bmal1 ‐ a critical component of the molecular circadian clock ‐ produces arrhythmic and infertile animals. Female Bmal1 KOs show no detectable LH surge but normal pituitary responsiveness, suggesting hypothalamic dysfunction. Our study sought to characterize neuroendocrine function in these mice, particularly responsiveness to kisspeptin. Corroborating previous studies, we found no difference in the LH response to 1 μg/kg GnRH 10 minutes after administration between wildtype (WT) and Bmal1 KOs (LH increase of 2.61 ± 0.25 ng/μl vs 2.51 ± 0.50 ng/μl, n=6). Interestingly, Bmal1 KO female mice had a significantly increased response to 30 nmole kisspeptin after 10 minutes (LH increase of 1.19 ± 0.34 ng/μl vs. 3.08 ± 0.56 ng/μl, n = 6, p<0.05). In addition to having an increased LH response to a kisspeptin challenge, the knockout mice showed greater area under the curve in response to kisspeptin during a 45‐minute time course (38.99 ± 4.69 vs 163.30 ± 16.88 ng/min/μl, n=3–4, p<0.005). A dose response curve indicated no difference in ED50 (0.144 ± 0.048 vs. 0.085 ± 0.048 mg/kg, n=4) despite the increased maximal effect, suggesting kisspeptin is more efficacious in eliciting LH in Bmal1 knockout females. Since the sex‐specific expression of Kiss1 in the AVPV is critical for regulating ovulation in females, we hypothesized that this population may be absent in Bmal1 KO females. However, we found no differences in Kiss1 mRNA expression between WT and Bmal1 KO females (1.08 ± 0.25 vs 1.03±0.29‐fold change, n=4). Overall, our results suggest disruption of the hypothalamic and pituitary regulation of fertility in the Bmal1 KO females, specifically in responsiveness to kisspeptin. Our findings suggest that circadian cues are important for regulating the temporal release of hypothalamic peptides that regulate fertility, and these findings may help direct kisspeptin treatment in women with circadian‐derived hypothalamic amenorrhea.Support or Funding InformationNIH R01 HD072754, R01 HD082567, P50 HD012303, T32 HD007203
Increased α‐tubulin Acetylation Translocates Gαs out of Lipid‐rafts: a Novel Mechanism for Antidepressant Effects of HDAC‐6 Inhibitors
Authors: Singh Harinder M , Schappi Jeffrey , Rasenick Mark M 
Published: November 2023

Histone deacetylase‐6 (HDAC‐6) enzymes deacetylate α‐tubulin and have been shown to be upregulated during neuropsychiatric disorders. HDAC6 knockout or HDAC6 inhibitors displayed an antidepressant profile in animal models. While, a possible role for HDAC6 inhibitors in treatment of depression exists, the potential mechanism for this remains elusive. Previously, our group has shown that treatment of rats or C6 glioma cells with several classes of antidepressants result in facilitation of the G‐protein, Gαs, activity by translocating it from lipid‐rafts inducing a sustained elevation in cAMP production. Studies have also demonstrated interplay between tubulin and Gαs in lipid‐rafts. Once freed from lipid‐raft domains, Gαs, couples with adenylyl cyclase (AC). Although Gαs interacts directly with tubulin to modify microtubule dynamics, tubulin also acts as an anchor for Gαs in lipid rafts. Based on HDAC‐6 roles in tubulin deacetylation and our data showing Gαs complexes with tubulin in lipid‐rafts, we hypothesized that acetylation of α‐tubulin disrupts tubulin‐Gαs anchoring, rendering Gαs free to activate AC. To test this, C6 glial cells were treated with the HDAC‐6 inhibitor, tubastatin‐A. Three‐day treatment with tubastatin‐A not only increased acetylation of α‐tubulin but also caused translocation of Gαs out of lipid‐rafts. Fluorescence Recovery After Photobleaching (FRAP) on C6 cells stably expressing GFP‐Gαs, was conducted and cells treated for three days with tubastatin‐A showed an “antidepressant signature”. Finally, sustained elevation of cAMP was revealed by increased cAMP response element binding protein (CREB) phosphorylation and increased expression of brain derived neurotrophic factor (BDNF). An increase in acetylated α‐tubulin prevented the formation of tubulin‐Gαs‐complexes in lipid rafts. This did not appear to be a mechanism shared with other classes of antidepressants. These findings suggest HDAC6 inhibition shows incomplete commonality with traditional antidepressants. Nonetheless, it is possible that compounds that decrease tubulin‐Gαs interactions by increasing acetylation of a α‐tubulin may show promise for antidepressant therapy.Support or Funding InformationNIH R01 AT 009169 (MMR) VA BX 001149 (MMR) AHA Postdoctoral Fellowship (HS)NIH T32 MH0667631 (JS)
High Concentration of Rosiglitazone Reduces Low‐density Lipoprotein Receptor‐Related Protein‐1 (LRP1) Levels Via Lysosomal Degradation in HepG2 Cells
Authors: Rondón‐Ortiz Alejandro Nico , Gugnani Kuljeet , Maher Timothy J. , Pino‐Figueroa Alejandro 
Published: November 2023

LRP1 is a transmembrane receptor involved in the internalization of molecules by either transcellular transport or lysosomal degradation via clathrin. PPARγ is a nuclear receptor that transcribes several target genes involved in cell differentiation, lipid metabolism and glucose homeostasis. PPARγ activation has been shown to upregulate LRP1 mRNA and protein levels in HepG2 cells. However, overactivation of this nuclear receptor has exhibited a reduction in LRP1 protein levels. The aim of this research was to evaluate the effect of rosiglitazone (RGZ), a known PPARγ agonist, on LRP1 protein levels in HepG2 cells. Additionally, the LRP1 protein reduction mechanism in RGZ treated‐cells studying PPARγ activation, ubiquitin proteasome system and lysosomal degradation were evaluated.HepG2 cells were treated with RGZ (0.003 – 30 μM) to show no increase on LRP1 protein levels as reported in the literature. However, cells incubated for 24 and 48 hours with 30 μM of RGZ reduced LRP1 protein levels by 40 and 90%, respectively. PPARγ antagonism and ubiquitin proteasome system inhibition were studied in RGZ treated‐cells demonstrating no effect on LRP1 reduction. Bafilomycin A1, a lysosome degradation inhibitor, increased LRP1 protein levels up to 4‐fold in RGZ treated‐cells.These results suggest that overactivation of PPARγ reduces LRP1 protein levels via lysosome degradation. Further experiments are required to evaluate the mechanism involved in the effect of RGZ on LRP1 protein expression.
Involvement of KCa2.3 Channels in Relaxation of Erectile Tissue Is Altered in Type 2 Diabetic Mice
Authors: Steffensen Simon Gabriel Comerma , Mogensen Susie , Beck Lilliana , Simonsen Ulf 
Published: November 2023

IntroductionActivation of endothelial small conductance calcium‐activated K+ channels (KCa2.3 or SK3) and intermediate conductance calcium‐activated K+ channels (IK or KCa3.1) leads to vascular relaxation. Our previous studies have shown that KCa2.3 down‐regulation diminishes the normal erectile function; this channel it is the most expressed KCa channels in the endothelium of corpus cavernosum. In the present study we hypothesized that KCa2.3 channel function is altered in erectile tissue of type 2 diabetic animals.Materials and MethodsErectile function was measured in diabetic db/db mice was compared to heterozygous control animals. Corpus cavernosum strips were mounted for isometric tension recording and processed for qPCR or Western Blot.ResultsIn anesthetized diabetic db/db mice erectile function was diminished compared to control animals. In isolated corpus cavernosum strips from db/db mice, concentration‐dependent contractions to noradrenaline were incremented compared to strips from control mice. Apamin, a blocker of KCa2 channels blocked acetylcholine relaxation in corpus cavernosum from db/db and control animals, being less effective in db/db compared to control animals. NS309 (0.5 μM), an activator of KCa2 and KCa3.1 channels, potentiated acetylcholine concentration‐dependent relaxations in corpus cavernosum from control, but this was not the case in corpus cavernosum from db/db mice. SNP relaxation was similar in corpus cavernosum from db/db and control animals. KCa2.3 gene and protein expression was incremented in corpus cavernosum from db/db mice, nevertheless protein expressed in the aorta was diminished.ConclusionsOur results suggest that in type 2 diabetes KCa2.3 channel expression is down‐regulated in the systemic circulation, and upregulated in erectile tissue. In erectile tissue noradrenaline contraction is increased and the effect of NS309 reduced which may be related to impaired KCa2.3 channel function. These changes may contribute to impairment of erectile function in diabetes.Support or Funding InformationWe acknowledge Aarhus University, Novo Nordisk and Consejo de Desarrollo Cientifico y Humanistico de la Universidad Central de Venezuela (CDCH‐UCV) for their support.
Thiol‐Oxidation Impairs Alpha2‐Adrenergic Receptor Mediated Inhibition of N‐type Ca2+ channels and Results in Receptor Mislocalization
Authors: Mui Ryan K , Fernandes Roxanne N , Feng Huijie , Galligan James J 
Published: November 2023

IntroductionOxidative stress and sympathetic hyperactivity contribute to hypertension. In sympathetic neurons, activation of alpha2‐adrenergic receptors (α2AR) decreases nerve terminal release of norepinephrine (NE) by activating Gαi/o proteins to inhibit N‐type voltage‐gated Ca2+channels. We have shown in DOCA‐salt hypertensive rats that α2AR‐mediated inhibition of Ca2+channels is impaired in sympathetic neurons, resulting in increased NE release. Furthermore, blood pressure and α2AR function are preserved following treatment with clodronate (macrophage depletion agent) and apocynin (NADPH oxidase inhibitor). Oxidative stress is associated with adrenergic dysfunction and Gαi/o are targets of oxidative stress. Inhibition of α2AR‐Gαi/o coupling following pertussis toxin (PTX) treatment occurs due to ADP‐ribosylation on the C‐terminal cysteine (position 351) of Gαi/o. It is not known if thiol‐oxidation of this residue results in similar uncoupling. Therefore, we tested the hypothesis that oxidizing agents impair α2‐AR function by thiol‐oxidation of Cys351 on Gαi/o.MethodsThese studies used cultured sympathetic neurons, G1A1 cells (HEK cells stably expressing N‐type Ca2+ channels), and HEK 293T cells. Cell lines were co‐transfected with α2AR and either WT‐Gαo or PTX‐insensitive Gαo (PTXi‐Gαo, Cys351Gly). Oxidative stress was induced with H2O2, t‐BOOH, or diamide (thiol‐specific oxidant). Whole cell patch clamp was used to detect inhibition of Ca2+ current using NE to activate α2AR and GppNHp to inhibit Ca2+ current independent of α2AR activation. Immunohistochemistry was used to localize α2AR and Gαo following oxidant treatment before and after activation of α2AR with NE.ResultsIn sympathetic neurons, α2AR‐mediated inhibition of Ca2+ current was reduced following H2O2, t‐BOOH or diamide treatment vs. untreated neurons. GppNHp inhibited Ca2+ current equally in all neurons independent of oxidative stress. Oxidative stress impaired α2AR‐mediated inhibition of Ca2+ current similarly in G1A1 cells co‐transfected with WT‐Gαo or PTXi‐Gαo. Oxidative stress had no effect on cell capacitance or Ca2+ channel activity in neurons and G1A1 cells. In HEK 293T cells, oxidative stress did not affect co‐localization of α2AR and Gαo, however, oxidative stress resulted in greater cytoplasmic localization of α2AR.ConclusionsThiol‐oxidation results in impaired α2AR‐mediated inhibition of Ca2+ current. Thiol‐oxidation does not affect Gαo coupling to α2AR. Dysfunction may be attributed to thiol‐oxidation of cysteine residues on α2AR, which do not affect Gαo coupling, but may alter structural motifs that could affect receptor localization to the cell membrane.Support or Funding InformationSupported by NIH 2P01HL070687 and American Heart Association (15PRE25090218)
ADP Ribosylation Factor 6 (Arf6) Modulates the Cell Surface Expression of the Cardiac Acetylcholine‐Activated Inward Rectifier Potassium Current (IKACh) Proteins
Authors: Alhadidy Mohammed M , Reiser Michelle , Szekeres Charles , Cha Byeong , Noujaim Sami F. 
Published: November 2023

IntroductionIt has been suggested that the ADP ribosylation factor 6 (Arf6) plays a role in the trafficking of Kir3.1 and Kir3.4 proteins ‐ the molecular correlates of the cardiac acetylcholine‐activated inward rectifier potassium current (IKACh). However, further details need to be elucidated. Therefore, we investigated the effects of Arf6 activity on the localization of Kir3.1 and Kir3.4 proteins using flow cytometry, Arf6 activation pull‐down assay, and live confocal microscopy. We tested the hypothesis that Arf6 cycling between the GDP‐ and GTP‐bound forms modulates the cell surface expression of IKACh proteins.MethodsStudies were performed in a human embryonic kidney (HEK293) cell line stably expressing Kir3.1 and Kir3.4 (GIRK cells). The IKACh proteins at the cell surface were labelled with TertiapinQ‐ATTO‐488 and quantified using flow cytometry in GIRK cells transiently transfected with Arf6WT (wild type), Arf6T27N (dominant negative, GDP‐bound form), or Arf6Q67L (constitutively active, GTP‐bound form). Additionally, GIRK cells were treated for 30 minutes with 10 μM Chloroquine or Primaquine, then cell surface IKACh proteins were quantified using flow cytometry, and their internalization was studied using live confocal microscopy. Arf6 activation by Chloroquine was assayed using purified Arf6 protein and human atrial tissue samples in pull‐down experiments. Results are reported as average ± standard error.ResultsIn flow cytometry experiments, both Arf6T27N (dominant negative) and Arf6Q67L (constitutively active) showed significant increase in IKACh proteins expression at the cell surface compared to Arf6WT (Arf6T27N, 14% ± 3, p<0.01; Arf6Q67L, 28.5% ± 4, p<0.01). This suggested that interference with Arf6 cycling between the GDP‐ and GTP‐bound forms leads to cell surface accumulation of the IKACh proteins. Moreover, treatment of GIRK cells with 10 μM Chloroquine for 30 minutes significantly decreased cell surface IKACh proteins (8% ± 1.7, p<0.01). Conversely, treatment with Primaquine, a structurally similar 4‐aminoquinoline, did not show the same effect. In live confocal microscopy studies, Chloroquine promoted the internalization of IKACh proteins compared to control. Arf6 activation pull‐down experiments using purified Arf6 protein and human atrial tissue samples suggested that Chloroquine, but not Primaquine, increased Arf6 activity.ConclusionExpression of the IKACh proteins at the cell surface is, in part, modulated by an Arf6‐dependent pathway. Constitutive interference with Arf6 cycling between GDP‐ and GTP‐bound forms increases the cell surface density of IKACh proteins, while transient pharmacological activation of Arf6 could lead to their internalization.Support or Funding InformationFunded in part by NIH Grant R01HL129136 to SFN
Pannexin‐1 Channel Activity and Associated ATP Release in Podocytes in Response to Adipokines
Authors: Li Guangbi , Zhang Qinghua , Li Ningjun , Ritter Joseph K. , Li Pin‐Lan 
Published: November 2023

Pannexin‐1 (PANX1) channel has been reported to mediate the release of ATP that is involved in local tissue inflammation, obesity, and many chronic degenerative diseases. It remains unknown whether PANX1 channel is present in podocytes and thereby mediates ATP release leading to glomerular inflammation or fibrosis. In the present study, we characterized the PANX1 channel in podocytes and examined the possibility of this channel in mediating ATP release induced by adipokines. By real time RT‐PCR, Western blot analysis and immunohistochemistry, we demonstrated that among 3 different pannexins, PANX1 is mostly enriched in podocytes either cultured or in situ in mouse glomeruli. Using patch clamp technique, we recorded a large voltage‐gated outward current under the Cs+in/Na+out gradient, which was 290.2±8.7 pA at +80 mV. Na‐gluconate or Na‐asparate substitution for NaCl in the bath solution blocked voltage‐gated outward currents and shifted the reversal potential of PANX1 currents to right, indicating the anion permeability of this channel. Pharmacologically, the recorded voltage‐gated outward currents were substantially attenuate by either carbenoxolone (CBX) or probenecid (PBNC), the specific PANX1 channel inhibitors (to 23.8±2.5% and 21.4±1.7% of control, respectively). Given the stimulatory role of extracellular ATP in inflammation and chronic low‐grade inflammation as a hallmark of obesity, we tested whether adipokines from fat cells activate PANX1 channel activity to release ATP. Adipokine, visfatin was shown to remarkably enhance pannexin‐1 channel activity in podocytes (to 252.5±7.7% of control), which was completely abolished by CBX. However, another adipokine, adiponectin significantly blocked pannexin‐1 channel activity to 29.9±4.4% of control, a level similar to the effects of CBX and PBNC. Correspondingly, visfatin significantly increased ATP release from podocytes by almost 2 folds, which was prevented by CBX, PBNC or adiponectin. These results suggest that PANX1 functions as an anion channel in podocytes to control ATP release, which is regulated by adipokines.Support or Funding InformationThis study is supported by NIH grants DK54927, DK102539, HL057244, HL075316, and HL122937.