bluuebunny/crossref_metadata_2025 huggingface dataset:
Persistent Inflammation Induces Desensitization of the Presynaptic Cannabinoid 1 Receptor in the Ventrolateral Periaqueductal Gray
Authors: Bouchet Courtney A. , Eshleman Amy J. , Janowsky Aaron , Ingram Susan L.
Published: May 2022

The cannabinoid system is highly plastic and sensitive to environmental perturbations, often leading to downregulation of the cannabinoid 1 receptor (CB1R). Here, we investigate adaptations in CB1R regulation after persistent inflammation using patch clamp electrophysiology and radioligand binding. Under basal conditions, activation of the presynaptic CB1R with the cannabinoid receptor agonists WIN‐55,212‐2 or CP‐55,940 suppress GABA release. This suppression is reversed by the CB1R‐selective antagonists SR141716A (rimonabant) or AM251. After 5‐7 days of inflammation induced by intraplantar injection of Complete Freund’s Adjuvant (CFA), CB1R function is significantly reduced. Interestingly, radioligand binding assays indicate that the receptor binding is not different between naïve and CFA‐treated animals. Our data indicate that in the ventrolateral periaqueductal gray, persistent inflammation causes desensitization, as opposed to downregulation, of the CB1R in both male and female Sprague Dawley rats. Further, we find that persistent inflammation increases endocannabinoid levels, potentially leading to prolonged activation of the CB1R and desensitization. These data highlight an important regulatory mechanism for the CB1R in response to persistent inflammation.
A Role for Cortical Dopamine in the Paradoxical Calming Effects of Psychostimulants
Authors: Urs Nikhil M. , Harris Sharonda , Green Sara M. , Kumar Mayank M.
Published: May 2022

Attention deficit hyperactivity disorder (ADHD) affects children and adults, and manifests symptoms such as hyperactivity, impulsivity and cognitive disabilities. Psychostimulants, which are the primary treatment for ADHD, target monoamine transporters and have a paradoxical calming effect, but their mechanism of action is unclear. Studies using the dopamine (DA) transporter (DAT) knockout mice, which have elevated striatal DA levels and are considered an animal model of ADHD, have suggested that the paradoxical calming effect of psychostimulants might be through the actions on serotonin neurotransmission. On the other hand, newer non‐stimulant class of drugs such as atomoxetine and guanfacine suggest that targeting the norepinephrine (NE) system in the PFC might explain this paradoxical calming effect. We sought to decipher the mechanism of this paradoxical effect of psychostimulants through an integrated approach using ex vivo monoamine efflux experiments, monoamine transporter knockout mice, drug infusions and behavior. Our ex vivo efflux experiments reveal that NE transporter (NET) blocker desipramine elevates both norepinephrine and dopamine but not serotonin levels, in PFC tissue slices from wild‐type and DAT‐KO but not NET‐KO mice. However, serotonin (5‐HT) transporter (SERT) inhibitor fluoxetine elevates only serotonin in all three genotypes. Systemic administration of both desipramine and fluoxetine but local PFC infusion of only desipramine and not fluoxetine inhibits hyperactivity in the DAT‐KO mice. In contrast, pharmacological norepinephrine depletion but dopamine elevation using Nepicastat also inhibits hyperactivity in DAT‐KO mice. Together, these data suggest that elevation of PFC dopamine and not norepinephrine or serotonin as a convergent mechanism for the paradoxical psychostimulant effects observed in ADHD therapy.
Assessing the Effect of Organic Anion Transporter Inhibition on Circulating Pyridoxic Acid, an Endogenous Transporter Biomarker, using Physiologically‐based Pharmacokinetic Modeling
Authors: Thakur Aarzoo , Prasad Bhagwat
Published: May 2022

Transporter‐mediated renal secretion plays a critical role in the systemic clearance of ~30% of the Food and Drug Administration (FDA)‐approved drugs and is attributed to clinically significant drug‐drug interactions (DDIs). For example, organic anion transporters (OATs) are inhibited by perpetrator drugs (e.g., probenecid), leading to higher circulating levels of OAT substrates (e.g., furosemide).1 Recently, OAT inhibition has been shown to be associated with increased plasma levels of endogenous substrates, e.g., pyridoxic acid (PDA) and homovanillic acid, which can serve as potential OAT biomarkers.2 The objective of our study was to develop and validate a physiologically‐based pharmacokinetic (PBPK) model of PDA to allow prospective evaluation of the magnitude of potential DDI of an OAT inhibitor‐substrate pair. Probenecid and furosemide were used as selective inhibitor and substrate of OATs, respectively. Physicochemical properties and in vitro data pertaining to the pharmacokinetics of probenecid and PDA were collated from literature. A PBPK model was first constructed to predict the plasma concentration‐time profiles of probenecid after oral administration using Simcyp (v20). Then, utilizing the fraction secreted versus filtered as well as the unbound fraction (fu = 0.092) data of PDA,2 the total elimination and synthesis rates of PDA at steady‐state were used to develop a baseline PBPK model. The effect of OAT inhibition by probenecid on the PDA levels was estimated and the predicted area under the plasma concentration‐time profile (AUC), change in clearance, and the highest plasma concentration (Cmax) values were compared with the observed data.2 The model was able to capture the changes within 2‐fold of the clinical data. Using the predicted AUC profile of PDA and assuming 100% inhibition at Cmax, the magnitude of inhibition was estimated across different time points. The biomarker‐informed magnitude of inhibition showed significant correlation (r2 = 0.9336) with the observed change in furosemide concentrations in the presence of probenecid (Fig. 1). PDA‐informed PBPK model developed here can be used to prospectively predict the magnitude of OAT inhibition, which is particularly applicable to assessing DDI potential of investigational drugs or prescribed drugs in special populations with sparse samples, without the requirement of an exogenous probe substrate.

References:

1. Smith, D. E. et al. (1980). Journal of Pharmaceutical Sciences, 69, 571.

2. Shen, H. et al. (2019). Journal of Pharmacology and Experimental Therapeutics, 68, 136.
Altered GABAergic neurotransmission at the nucleus tractus solitarii following Hindlimb Unloading
Authors: Lima‐Silveira Ludmila , Hasser Eileen M. , Kline David D.
Published: May 2022

Hindlimb Unloading (HU) in rats induces dysautonomia and cardiovascular deconditioning (CVD) similar to that seen in bedridden individuals. The autonomic alterations in CVD and HU are related to plasticity within the central nervous system. The nucleus tractus solitarii (nTS) is a major integrative brainstem region for autonomic modulation and processing of cardiovascular reflexes. GABA and glutamate are the main inhibitory and excitatory neurotransmitters within this nucleus. HU increases nTS glutamatergic neurotransmission yet decreases neuronal excitability. In this study, we investigated the effects of HU on the GABAergic neurotransmission in the nTS. We hypothesized that HU increases GABA synaptic modulation of nTS neurons, which contributes to impaired autonomic and cardiovascular control. HU was induced by placing stainless wire rings in the tail of male Sprague‐Dawley rats (3 weeks old), and after one week of recovery suspending rats at an angle of 30–35° for two weeks with free access to food and water. Control animals were maintained in normal postural conditions. After HU or the control period, coronal brainstem slices were generated and whole‐cell patch clamp was performed to evaluate GABAergic neurotransmission in the nTS region. The magnitude of GABA release was evaluated via an intensity‐based GABA sensing fluorescence reporter (iGABASnFR). Block of GABAA receptors (Gabazine, 20µM and Bicuculline, 20µM) depolarized membrane potential in HU but not in Control, suggesting greater tonic GABA inhibition in HU. In response to GABA interneuron stimulation the evoked IPSC amplitude and area, as well as iGABASnFR fluorescence, was greater in HU than Control. HU also elevated the frequency of spontaneous miniature IPSCs but not their amplitude. Puffer application of GABA (100µM, 15ms) produced similar postsynaptic current responses in nTS neurons of HU and Control. These results indicate that HU increases GABAergic signaling in the nTS likely via augmented release of GABA from presynaptic terminals. This synaptic plasticity may be a component of the mechanisms underlying the autonomic and cardiovascular alterations following CVD.
A CURE for What Ails You: Relating Current Events to New Research in a Course‐Based Undergraduate Research Experience (CURE) in Physiology
Authors: Keesom Sarah M. , Provost Terri M.
Published: May 2022

The American Association of Colleges and Universities has identified participation in research as a high impact practice for undergraduate education. Course‐based undergraduate research experiences (CUREs) offer an excellent opportunity for involving the most students in research, especially students who might not be able to participate in research activities outside of class. Despite the benefits, the integration of a CURE in an animal physiology laboratory course can be a challenge when working with vertebrate animals, in part due to the opposing goals of reinforcing scientific skills (e.g., statistical analysis, which requires sufficient sample sizes) versus reducing the total number of research animals. We therefore designed an undergraduate animal physiology laboratory course to test the idea that a themed CURE allows physiology students to practice their scientific skills and produce publishable results within the context of an authentic research project, while decreasing the number of animals used for the course as a whole. To do this, we chose the theme “social isolation and physiology” to contextualize the research within current events and engage students’ curiosity about how social distancing measures necessitated by the COVID‐19 pandemic may influence physiology. To conduct this CURE, a class experiment was designed using 18 female laboratory mice, with 9 female mice in individual housing and 9 female mice in group housing. We then allowed students to develop their own hypotheses and methods to investigate the effects of social isolation on a physiological variable of their choice, using the class set of experimental animals. We used an iterative course design to provide students with the feedback they needed in order to be successful, which included multiple scaffolding assignments, as well as weekly one‐on‐one meetings to discuss their research project with their laboratory instructor. As final products of this project, students gave an oral presentation of their research findings and wrote a paper in manuscript format as would be suitable for scientific publication. At the end of the semester, we administered two surveys (one open‐ended and one Likert‐scale) to assess the impact of this theme‐based CURE on students. Our preliminary findings indicate that this CURE was the first authentic research experience for the majority of our students. Survey responses suggest that students were better able to conceptualize the research process after participating in the CURE and that students viewed themselves as scientists. Furthermore, responses from students highlighted that having the opportunity to face setbacks and troubleshoot problems was a new, but beneficial experience. Taken together, our findings indicate that a themed CURE is a viable model for integrating research experiences into upper level animal physiology laboratory courses.
Prenatal exposures to electronic‐cigarette aerosols affect fetal lung development in mice
Authors: Noel Alexandra
Published: May 2022

In the United States, there is an increasing number of teenagers and young adults, including women of childbearing age, who vape. With more than 10% of women admitting using electronic‐cigarettes (e‐cigs) during their pregnancy, there is a pressing need to investigate the effects of in utero e‐cig aerosol exposures on the developing lungs. As shown with active smoking during pregnancy, nicotine can alter prenatal development. Since e‐cigs are nicotine delivery devices, we used mouse models of in utero exposures to further our understanding of how flavored e‐cig aerosols affect the developing lungs, both at birth and later in life.
One Assay, Two Vital Values‐Estimating Intrinsic Permeability and Efflux Using MDCK‐MDR1 Assay
Authors: Zang Grace , Li Chun
Published: May 2022

Background

Intestinal intrinsic permeability and transporter mediated efflux are two major determinants of oral absorption. Typically, wild type Madin‐Darby canine kidney cells (MDCK) and MDCK cells transfected with the human MDR1 gene (MDCK‐MDR1) are used to evaluate intrinsic permeability and efflux, respectively. The MDCK assay provides intrinsic permeability but lacks transporter mediated efflux. Conversely, the MDCK‐MDR1 assay provides efflux ratio and “apparent permeability”. However, the apparent permeability can differ significantly from intrinsic permeability due to efflux. Traditionally, both assays (MDCK, MDCK‐MDR1) are applied separately and sequentially, which can be time‐consuming and costly.

In this study, we present a novel approach to estimate intrinsic permeability and efflux simultaneously using the MDCK‐MDR1 assay alone. Intrinsic permeability can be calculated using a scaling method by combining the 3 parameters (bi‐directional apparent permeability values and efflux ratio) determined from the MDCK‐MDR1 assay.

Methods

Test compounds of varying permeability and efflux ratios were used to assess the validity of the hypothesis. Results from the MDCK, MDCK‐MDR1 and caco‐2 assays were analyzed. Efflux ratio is estimated by dividing the apparent permeability from the basolateral to apical direction (PappBA) by the apparent permeability from the apical to basolateral direction (PappAB). Different averaging methods were used to calculate intrinsic permeability from MDCK‐MDR1 assay, and the measured intrinsic permeability from the MDCK assay was used as a comparison to find the most accurate averaging method.

Results

Of the different scaling and averaging methods used for estimating intrinsic permeability from MDCK‐MDR1 assay, the best method we found was a “quotient” scheme where calculated intrinsic permeability was adjusted based on the magnitude of efflux ratio. With this adjustment, both the intrinsic permeability and efflux ratio can be reliably obtained from the MDCK‐MDR1 assay for compounds with a wide range of efflux ratios. The calculated intrinsic permeability from the MDCK‐MDR1 assays aligns within 2 fold of the permeability values determined from the MDCK assay. Results from the caco‐2 cell line were also evaluated and applicable to the method mentioned above. Several hypotheses were formulated regarding why the “quotient” method was a better fit than simple apparent permeability averaging.

Conclusion

The strategy presented here provides an efficient and effective method to estimate both intrinsic permeability and efflux solely based on the MDCK‐MDR1 assay. Compared to the traditional approach of using MDCK and MDCK‐MDR1 assays separately, the method used here requires less time, cost, and compound for testing. This is particularly attractive for early drug discovery screening needs.
Immune cells play a crucial role in the development of a cardiac dysfunction in cardiac p38 MAPKa KO mice after pressure overload
Authors: Hemmers Anne , Nederlof Rianne , Kalfhues Lisa , Bottermann Katharina , Oenarto Vici , Gödecke Axel
Published: May 2022

Question

Cardiac metabolic remodeling is one of the early changes driving the progression of heart failure and is present even before overt cardiac dysfunction. Previously we showed that the tamoxifen inducible deletion of cardiomyocyte specific p38 MAPKa (KO) leads to a pronounced left ventricular dilation with a strongly impaired heart function just within 2 days of angiotensin II (ANGII) treatment. Additionally, a strong infiltration of immune cells in the cardiac tissue can be observed.

Methods and Results

Under baseline conditions no impairment of the heart function in KO mice can be observed. ANGII treatment for 2 days induces a strong left ventricular dilation and poor heart function, indicated by a reduced ejection fraction and an increase in end‐systolic and end‐diastolic volume. KO mice do not get used to constant ANGII treatment even after 7 days of ANGII treatment and cardiac function continues to deteriorate. KO mice transiently loose weight as a consequence of ANGII treatment. On the fourth to fifth day of ANGII treatment, KO animals gain weight again, although the heart function continues to deteriorate. FACS analysis of cardiac tissue revealed a strong infiltration of neutrophils in KO hearts after 48h of ANGII treatment (4.5‐fold increase compared to control hearts). A strong correlation between reduced ejection fraction and increasing amount of neutrophils in the cardiac tissue suggests a particularly important role of neutrophils in the development of cardiac dysfunction. Also an increase of macrophages (1.6‐fold increase), CD4+ T‐helper cells (2.5‐fold increase) and CD8+ cytotoxic T‐cells (2.5‐fold increase) as well as B‐cells (2‐fold increase) could be shown in KO hearts compared to control hearts. After long term ANGII treatment for 7 days, however, the number of neutrophils in the hearts decreased (10‐fold decrease in control hearts and 73‐fold decrease in KO hearts compared to 48h of ANGII treatment) and a strong increase in M2‐like macrophage infiltration, mainly in control hearts (1.6‐fold increase compared to 48h ANGII treatment), could be observed. The infiltrated macrophages were also positive for CCR2, which indicates they originated from monocytes. B‐ and T‐cells were still elevated in KO hearts after 7 days of ANGII treatment. Also in control hearts an increase of B‐ and T‐cells could be observed. In addition, fibrosis developed in the cardiac tissue, which could be shown by histological staining of collagen I and FACS analysis targeting αSMA+ cardiac fibroblasts.

Conclusion

Immune cells and in particular neutrophils play an important role in the development of cardiac dysfunction after ANGII induced pressure overload in p38 MAPKa KO mice. Over time, however, the number of immune cells in control mice also increases, which underlines the peculiarity of the first neutrophil influx after 48h ANGII treatment for the impairment of cardiac function.
Female Mice Exposed to Early Life Stress Display Exacerbated Obesity‐Hypertension while Normal Response to Adipose Afferent Reflex Stimulation
Authors: Dalmasso Carolina , Leachman Jacqueline R. , Ghuneim Sundus , Ahmed Nermin , Clifton Hollie , Loria Analia
Published: May 2022

The experimental stimulation of afferent excitatory signals from adipose tissue has been shown to contribute with obesity‐induced hypertension as part of a mechanism called the adipose afferent reflex (AAR). Previous studies from our lab have shown that male mice exposed to maternal separation and early weaning (MSEW), a mouse model of early life stress, display increased mean arterial pressure (MAP) and sympathetic tone compared to control mice when fed a high fat diet (HF). Also, MSEW‐HF males display exacerbated blood pressure responses to the acute stimulation of the AAR in perigonadal white adipose tissue (gWAT) compared to controls that correlates with increased neuronal activation in the posterior paraventricular nucleus of the hypothalamus and rostroventrolateral medulla. We have identified increased serotonin (5‐HT) levels in eWAT from obese MSEW mice as a potential endogenous factor stimulating sensory neurons. As females MSEW do not show sympathetic activation, the aim of this study was to determine whether their increased blood pressure was independent of exacerbated blood pressure response to AAR stimulation or levels of 5‐HT in gWAT. C57BL/6J mice pups were separated from the dams from postnatal day (PD) 2 to PD 16 and weaned early on PD 17. Control (C) litters remained undisturbed with the dams and were weaned on PD 21. After weaning, Female C and MSEW mice (n=5/group) were placed on low fat diet (LF) or HF (10% and 60% Kcal from fat, respectively). At 16 weeks, subcutaneous (sc) and gWAT depots were exposed for vehicle or 1.5 pmol/μL of capsaicin (8 μL, 2’, 4 sites, bilateral). Vehicle infusions in scWAT or gWAT did not change MAP from baseline in any of the groups. Capsaicin infusions in scWAT slightly increase MAP similarly in all groups (Delta MAP, mmHg: C‐LF: 3±1; MSEW‐LF: 3±1; C‐HF: 2±1; MSEW‐HF: 2±1). In gWAT, capsaicin infusions also increased MAP similarly in all groups (Delta MAP, mmHg: C‐LF: 3±1; MSEW‐LF: 4±1; C‐HF: 3±1; MSEW‐HF: 5±2). None of these changes in MAP were significant. No differences in 5‐HT levels in scWAT (C‐HF: 1801.15±122.26; MSEW‐HF: 1799.17±216.58 ug/mg tissue) and gWAT (C‐HF: 563.51±71.41; MSEW‐HF: 518.31±99.73 ug/mg tissue); were found between groups. Furthermore, stimulation of AAR with 5‐HT (10‐6 M, 4 sites, bilateral) increased blood pressure only in male MSEW mice (p<0.05). These results suggest that the AAR is not exacerbated in obese female MSEW mice, result that is consistent with a lack of sympathetic activation compared to control mice.
Contribution of Beta‐amyloid Accumulation to Cerebral Hypoperfusion in Alzheimer's Disease
Authors: Fang Xing , Zhang Huawei , Border Jane J. , Rivers Patrice L. , Strong Luke B. , Cooper Jonita , Crumpler Reece F. , Thomas Kirby N. , Roman Richard J. , Fan Fan
Published: May 2022

Alzheimer’s Disease (AD) is an emerging global health care crisis. However, underlying mechanisms are not understood well enough to translate to precision medicine. There is increasing evidence suggesting that AD is associated with brain hypoperfusion. However, it is unclear whether amyloid‐beta (Aβ) accumulation is a cause or consequence of AD, and how it contributes to cerebral hypoperfusion. The present study examined if Aβ accumulation induces cerebral hypoperfusion in AD by affecting cerebral vascular function via both anterograde (arteriole‐to‐capillary) and retrograde (capillary‐to‐arteriole) pathways in the TgF344‐AD rat model of Alzheimer's disease. We first confirmed that AD rats displayed hippocampal‐based cognitive dysfunction at 6 months of age using an eight‐arm water maze. We then found that AD rats exhibited impaired myogenic response (MR) of middle cerebral arteries (MCAs) and penetrating and parenchymal arterioles (PAs) two months earlier than the onset of cognitive deficits using a Living System pressure myograph. AD rats displayed poor surface and deep cortical cerebral blood flow (CBF) autoregulation recorded by laser Doppler flowmetry, and reduced functional hyperemic response induced by whisker stimulation. Moreover, cell contractile capabilities, detected by collagen gel based‐cell contraction kit, were reduced in Aβ‐treated cerebral VSMCs isolated from F344 rats, similar as seen in VSMCs isolated from AD rats. Furthermore, we found that the productions of reactive oxygen species (ROS) and mitochondrial superoxide in cerebral VSMCs isolated from AD rats were elevated using DHE staining and MitoSOX staining. Moreover, AD cells exhibited reduced mitochondrial respiration and ATP production detected by the Seahorse Cell Mito Stress Test kit. AD cerebral VSMCs also exhibited disrupted actin cytoskeleton and contractile units utilizing immunohistochemistry. Oxidative stress, mitochondrial dysfunction, and actin cytoskeleton disorganization are all factors that are associated with the reduced contractile capabilities of cerebral VSMCs mediated MR and CBF autoregulation. In other studies, we found that capillary endothelial cell‐derived inward rectifier potassium (Kir2.1) activity, which is responsible for retrograde CBF regulation, was reduced in the brain of AD rats using Western blot. PAs with capillaries isolated from AD rats dilated to a lesser degree than WT rats in response to moderately elevated extracellular K+ (10 mM) applied to capillaries. Inhibition of Kir2.1 channels with ML133 diminished the vasodilatory response to a greater extent in WT rats. These findings indicate that Aβ accumulation is associated with cerebral hypoperfusion in AD by affecting cerebral vascular function via both anterograde and retrograde pathways and provide novel insight into the vascular contribution to AD.
Amphotericin A21 Induces Cytotoxic Effects in Breast Cancer Cell Lines
Authors: Escobar‐Reséndiz Rubi , Ortega‐Blacke Ivan , Galván Arturo , Rodriguez‐Fragoso Lourdes
Published: May 2022

INTRODUCTION

Cancer is a public health problem that causes enormous loss of human life. Worldwide, breast cancer is one of the most diagnosed types of cancer and one that contributes significantly to cancer mortality. One of the situations that contributes to mortality statistics is that the patient may present low efficacy to treatment or the development of resistance. Currently there are various antineoplastic drugs, but these are not enough to face the complications of the disease. Therefore, it is of interest to study new drugs that have antineoplastic potential. Amphotericin B L‐histidine methyl ester, known as Amphotericin A21 (AmB‐A21), is a compound that has been extensively studied by our research group. Toxicological studies have shown that AmB‐A21 is a very safe compound, causing little toxicity in in vivo models. AmB‐A21 has also been found to have a cytotoxic effect, induce apoptosis, and reduce the proliferation and invasion of lung cancer cells (A549 cells). The results of the evaluations suggest that AmB‐A21 has the potential to be an effective and safe antineoplastic drug. The present project aims to evaluate whether AmB‐A21 can also cause antineoplastic effects in breast cancer cell lines, first evaluating the cytotoxic effect of AmB‐A21.

AIM

To evaluate the cytotoxic effect of AmB‐A21 on MDA‐MB‐231 and MCF7 cells.

HYPOTHESIS

AmB‐A21 induces cytotoxicity in MDA‐MB‐231 and MCF7 cells.

Methods

MDA‐MB‐231 (ATCC HTB‐26) and MCF‐7 (ATCC HTB‐22) lines from breast cancer were used. Both lines were cultured at 37 ° C, with 5% CO2, in DMEM HG medium supplemented with non‐essential amino acids, glutamine, sodium pyruvate, gentamicin and 10% fetal bovine serum.

First, growth kinetics were performed for 72 hours (h), to characterize the doubling rate and time of each cell line. Subsequently, dose‐response curves were performed to evaluate the cytotoxic effect of AmB‐A21 and inhibitory concentrations 50 (IC50) were calculated. Finally, the type of cell death that AmB‐A21 induces at IC25 y 50 was identified in MCF‐7 cells.

RESULTS

The proliferation rate at 72 h was found to be 2.43 ± 0.38 and 3.10 ± 0.21 for MDA‐MB‐231 and MCF‐7 cells respectively. Regarding the dose‐response curves, it was found that AmB‐A21 induces a concentration‐dependent cytotoxic effect in both breast cancer cell lines, whose IC50 were 178.83 ± 18.8 and 207.24 ± 5.7 µM/mL for the MDA‐MB‐231 and MCF‐7 cells, respectively. The results obtained had a statistically significant difference, with respect to the control. Data are shown as mean ± standard deviation. AmB‐A21 was also found to induce apoptosis cell death in MCF‐7 cells with their IC50 & 25.

CONCLUSIONS

AmB‐A21 induces cytotoxicity in cell lines originating from breast cancer (MDA‐MB‐231 and MCF‐7) and generates cell death by apoptosis in MCF‐7 cells.
Pharmacological Evaluation of Beta‐Hydroxifosphocarnitine in No‐Alcoholic Esteatohepatitis Induced in Rats
Authors: Sanchez‐Quevedo Janet , Rosado Jorge Luis , Rodriguez‐Fragoso Lourdes
Published: May 2022

Aim

Evaluate the pharmacological effect of Β‐Hydroxyphosphocarnitine (β‐HFC) on non‐alcoholic steatohepatitis induced in rats.

Introduction

Nonalcoholic steatotepatitis (NASH) is a serious global public health problem, it is related to other pathologies with increasing prevalence, manifested in the increased prevalence of NASH. Type 2 diabetes mellitus and obesity are the main pathologies related to NASH. There is no specific drug for NASH, the current drugs used for NASH are related to serious side effects, which is why there is an urgent need in the development of an effective therapy for NASH but with fewer side effects. L‐carnitine has an effect on improving metabolic alterations related to NASH, however, its intestinal absorption is limited. B‐HFC is an analog of L‐carnitine that has been shown to be safe for administration, to maintain pharmacological properties of its precursor and its absorption is greater than that of L‐carnitine. So it can be a good treatment for NASH.

Method

The NASH model was developed in male Wistar rats by ingesting carbonated drink as a source of fructose, coconut oil and carbon tetrachloride. 3 groups were formed with NASH (n = 8); NASH, NASH ‐β‐HFC and NASH‐pioglitazone. Another 3 groups of healthy rats (n = 8); control, control‐β‐HFC and control‐pioglitazone. The effect of β‐HFC in rats with NASH was evaluated by staining of histological sections of liver. The stains used were Masson's trichrome, hematoxylin and osin and Periodic Acid‐Schiff (PAS). Biochemical analysis was performed to evaluate the effect of β‐HFC on the metabolic alterations that characterize NASH.

Result

It was previously reported that β‐HFC reduced the triglyceride levels of rats with NASH, as well as the levels of liver enzymes. Evidencing the effect of β‐HFC in improving lipid metabolism, manifesting itself in less liver damage. For this occasion we will report the effect of β‐HFC on liver glycogen levels. Glycogen is a reserve of the cell, as a source of glucose in prolonged fasting states. Our results demonstrated a higher glycogen reserve in the NASH rats. This result was expected, since the rats had a high fructose intake. It is important to mention that the rats with NASH but treated with β‐HFC presented reduced glycogen levels, this effect can be attributed to a more efficient β‐oxidation process, which could also justify the reduction of triglycerides and fibrosis. Therefore, we could argue that a longer treatment time, the liver alterations in NASH would improve significantly.

Conclusion

β‐HFC has an effect on improving liver and metabolic alterations characteristic of the development of NASH.
Continuous Ultrasonic Transit‐Time Blood Flow and Arterial Blood Pressure Monitoring in Freely Moving Sprague‐Dawley Rats via Implantable Telemetry
Authors: Senador Danielle , Azar Toni , Fricke Kyle , Reynolds Christian , Minic Zeljka
Published: May 2022

BACKGROUND

Simultaneous blood flow and pressure measurements provide a more comprehensive hemodynamic assessment in cardiovascular studies. However, current cardiac output (CO) measurements, by flow probes or imaging, require either anesthesia or a tethered setup, which result in limitations due to anesthesia effects and/or monitoring time.

OBJECTIVE

The objective of this study was to use an implantable telemetry device (EndoGear (EG), Transonic Systems Inc., Ithaca, NY) to measure continuous CO (real‐time ascending aortic blood flow), and mean arterial pressure (MAP) in conscious, freely moving rats and to verify the ability of the system to capture well‐known circadian oscillations in major cardiovascular parameters.

METHODS

All procedures were approved by the Institutional Animal Care and Use Committee of Wayne State University and complied with the NIH Guide for the Care and Use of Laboratory Animals. Male Sprague‐Dawley rats were implanted with EG transmitters to continuously monitor CO, MAP, temperature, and activity for 5 weeks. An ultrasonic transit‐time flow probe was placed around the ascending aorta via a right lateral thoracotomy (4th intercostal space). The pressure catheter was inserted into the iliac artery and advanced (approximately 2.5 cm) into the abdominal aorta, while the transmitter was implanted intraperitoneally via a midline laparotomy approach. The inductive power receiver associated with the implant was positioned in an abdominal subcutaneous pocket. After surgery, rats received thermal support, prophylactic analgesics, and antibiotics, as well as fluid supplementation. Body weight, food and fluid intake were monitored daily for the first 10 days and then bi‐weekly until the end of the study. Animal checks and necessary handling were conducted at specific times to minimize circadian disturbances.

RESULTS

Changes in MAP, heart rate (HR, captured from pressure and flow traces), CO, stoke volume (SV, calculated as CO/HR), and temperature revealed, as expected, a clear circadian pattern characterized by lower values during the light phase. MAP decreased by ~5%, from 96.9 ± 0.15 to 92.1 ± 0.19 mmHg (p<0.05). HR decreased by ~10%, from 369 ± 3 to 329 ± 6 bpm (p<0.05). CO decreased by ~14%, from 91.6 ± 1.79 to 78.4 ± 1.89 ml/min (p<0.05). SV decreased by ~7%, from 245 ± 3.5 to 238 ± 1.4 μl (p<0.05). The decreases in temperature and activity during the light phase were subtle yet significant.

CONCLUSIONS

The telemetry device was able to effectively measure continuous changes in CO and MAP. Preservation of the circadian rhythm indicates favorable post‐operative outcome after extensive surgery and complex chronic implantation in rats.
Activation and fine‐tuning of Ca2+‐dependent slow afterhyperpolarizations by Ca2+‐handling organelles in vasopressin magnocellular neurosecretory neurons
Authors: Kirchner Matthew K. , Stern Javier
Published: May 2022

Vasopressin (VP) magnocellular neurons of the supraoptic nucleus become hyperexcitable in cardiovascular diseases such as heart failure. This hyperexcitability is caused by changes in both synaptic and intrinsic mechanisms, including the slow afterhyperpolarization (sAHP), a phenomenon underlain by a calcium‐dependent K+ current. While the general features of the sAHP and its effects on firing are well characterized, the precise spatiotemporal mechanisms of Ca2+ activation and buffering of the sAHP in VP neurons remains unknown. Combining patch clamp electrophysiology, live Ca2+ imaging, and photolytic Ca2+ uncaging in transgenic eGFP‐VP rats, we investigated the role of endoplasmic reticulum (ER) and mitochondria (MITO) in shaping sAHPs. We generated and measured sAHPs in current clamp via current injection or rapid targeted intracellular Ca2+ uncaging. Pre‐incubation of slices with thapsigargin (3μM) to deplete ER Ca2+ stores inhibited sAHPs significantly while leaving activity‐evoked Ca2+ transients unaffected. When sAHPs were evoked via Ca2+ uncaging, some sAHP remained, but was still significantly inhibited by thapsigargin. Additionally, acute caffeine mobilization of ER stores enhanced sAHP magnitude. These results demonstrate that ER Ca2+ release is necessary for sAHP activation in VP neurons. Conversely, blockade of MITO Ca2+ release via blocking MITO Ca2+ exchangers with TPP (20μM) failed to inhibit the sAHP. Conversely, blockade of mitochondrial Ca2+ buffering with bath application of the MITO uncoupler CCCP (1μM) or the MITO Ca2+ uniporter (MCU) blocker Ru360 (20μM) in the patch pipette significantly prolonged the decay time constant of both the sAHP and the underlying Ca2+ transient. These results demonstrate that the MCU regulates spatiotemporal dynamics of the sAHP. Combining thapsigargin with Ru360 showed an initial block of the sAHP, followed by an enhancement over 15 minutes as Ru360 dialyzed into the cell. Finally, targeted Ca2+ uncaging in the dendrites failed to generate an sAHP, providing insight into the morphological compartmentalization of sAHP channels. These results support the ER (but not MITO) as a major source of Ca2+ activating the sAHP, as well as a role for the MCU in regulating the magnitude and time course of the sAHP via Ca2+ buffering. Taken together, our studies support a fine‐tuned interaction between VGCCs, ER, and MITO in modulating the magnitude and time course of the sAHP in VP neurons. Future studies warrant investigation into whether changes in these interactions contribute to altered VP neuronal activity in cardiovascular disease states.
Eplerenone‐induced natriuresis and hyperkalemia in mice lacking aldosterone results from aldosterone‐independent mineralocorticoid receptor occupancy
Authors: McCormick James A. , Maeoka Yujiro , Sharma Avika , Su Xiao‐Tong , Wang Wen‐Hui , Ellison David H.
Published: May 2022

Background

Mineralocorticoid receptor (MR) antagonists are recommended for patients with resistant hypertension independent of circulating aldosterone levels. Aldosterone, secreted in response to salt depletion or hyperkalemia, activates MR to increase epithelial sodium channel (ENaC) activity. While glucocorticoids can also activate MR, they are metabolized by 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2), protecting MR from illicit glucocorticoid occupation. 11β‐HSD2 is expressed at increasing levels from the DCT through the collecting duct. Here, we hypothesized that glucocorticoid occupies MR to stimulate ENaC in the DCT2 and early connecting tubule.

Methods

We studied aldosterone synthase knockout (AS−/−) mice provided by Dr. JM Luther. Kidney–specific MR knockout (MR−/−) mice were used for validation of Western blot for ENaC and immunofluorescence for MR. We measured plasma and urinary electrolytes at baseline, and following dietary salt restriction with or without administration of the MR antagonist eplerenone.

Results

AS−/− mice displayed higher plasma K+ levels than AS+/+ mice but not salt wasting under baseline. Under low salt (LS) diet, AS−/− mice exhibited higher urine Na+ excretion, but preserved body weight. Cleaved αENaC and γENaC abundances were lower in AS−/− than AS+/+ mice, and were not increased in AS−/− mice after LS diet. Notably, we found nuclear localization of MR was preserved in DCT2 of AS−/− mice. Administration of the MR blocker eplerenone to AS−/− mice fed LS diet led to hyperkalemia and decreased body weight with higher Na+ excretion, mimicking the phenotype of MR−/− mice.

Conclusions

Our results provide evidence that MR is occupied in the absence of aldosterone, suggesting glucocorticoid‐binding to MR preserves sodium homeostasis in the DCT2 of AS−/− mice. This likely explains the milder phenotype of AS−/− mice compared with MR−/− mice.
Structural Enzymology of the Cysteine Tryptophylquinone (CTQ) Enzyme GoxA
Authors: Yukl Erik , Avalos Dante , Mamounis Kyle , Davidson Victor
Published: May 2022

GoxA from Pseudoalteromonas luteoviolaceae belongs to a recently identified family of enzymes (the LodA‐like proteins) that utilize a post‐translationally derived cysteine tryptophylquinone (CTQ) cofactor for catalysis. In contrast to other tryptophylquinone enzymes, GoxA acts as an oxidase rather than a dehydrogenase, using molecular oxygen to catalyze the oxidative deamination of glycine to produce ammonia, glyoxylate and hydrogen peroxide. The crystal structure of this enzyme revealed a homotetramer with a novel alpha helical domain and extensive inter‐subunit interactions, which account for the positive cooperativity observed toward its substrate by steady‐state kinetics. Soaking GoxA crystals in glycine results in a dramatic color change from yellow to blue, indicating the formation of an unusual intermediate at the CTQ site that was also detected in solution. The structure of this intermediate reveals a Schiff‐base formed between the substrate and CTQ that is stabilized by numerous hydrogen bond contacts with residues both in the active site and from a neighboring protomer. The kinetics and structures of active site mutants reveal the roles of specific residues in substrate binding, cooperativity, and catalysis, and allowed for the detection of another intermediate in the GoxA reaction pathway. This combination of kinetics, spectroscopy and X‐ray crystallography has allowed for the construction of a detailed reaction mechanism for this unusual enzyme with high‐resolution structures now available for key intermediates.
sFlt1‐Induced Preeclampsia Enhances Cardiovascular Response to Post Partum Hypertensive Stimuli via Smooth Muscle Mineralocorticoid Receptor
Authors: Biwer Lauren , Lu Qing , Ibarrola Jaime , Stepanian Alec , Man Joshua , Carvajal Brigett , Camarda Nicholas , Zsengeller Zsuzsanna , Seely Ellen , Karumanchi S. A. , Jaffe Iris
Published: May 2022

Background

Preeclampsia (PE), a syndrome of high blood pressure (BP) and renal damage in late pregnancy, is associated with increased soluble VEGF receptor (sFlt1) and survivors have increased risk of future hypertension and cardiovascular disease.

Hypothesis

Since smooth muscle cell mineralocorticoid receptor (SMC‐MR) can be activated by hypertensive stimuli, we hypothesized that high sFlt1 exposure during pregnancy may induce a post‐partum state of enhanced vascular sensitivity via SMC‐MR activation.

Methods/Results

A PE model was induced by transient viral expression of sFlt1 in pregnant C57Bl6 mice. Elevated serum sFlt1, BP and glomerular endotheliosis was confirmed which all resolve post‐partum. In a small cohort of women with prior PE, we confirm salt sensitivity of BP. Thus, postpartum mice were implanted with telemetric BP monitors and exposed to repeated hypertensive stimuli (high salt and subsequent AngII). Mice with prior PE had a significantly increased BP response to hypertensive stimuli. Microvascular arteries from mice after PE and hypertensive stimuli had enhanced ex vivomyogenic tone and AngII vasoconstriction. To test the direct impact of sFlt1 on SMC‐MR function, cultured SMC (Pac1) were transiently exposed to sFlt1 (24 hours on then off) to mimic the post‐PE state. SMC‐MR transcriptional activity in response to aldosterone and AngII were significantly increased after sFlt1 exposure as measured by luciferase reporter assay. Target gene expression was also enhanced after sFlt1 exposure. Finally, the role of SMC‐MR in the post‐PE phenotype was tested in vivo by PE induction in SMC‐MR‐KO vs MR‐intact littermates. Post partum, SMC‐MR‐KO mice were protected from the PE‐induced increase in aortic stiffness, microvascular myogenic tone and AngII vasoconstriction.

Conclusion

sFlt1‐induced PE produces a state of enhanced sensitivity to stimuli that may be mediated by increased activation of SMC‐MR. These data support the use of MR antagonists to mitigate the increased risk of cardiovascular disease in women exposed to PE.
A Computational Biology Approach to pH Selective Reactions in the Extracellular Fluid of Cancer Cells Based in the Gibbs Free Energy Minimization Approach
Authors: Colón Colón Carolina C. , Soto Acevedo Angeliz A. , Narváez Ramos Michelle A. , Malavé Darlene , Castro Rosario Miguel E.
Published: May 2022

A common denominator of many cancer cells is that the extracellular pHe value is acidic (pHe<7) in contrast to the basic pHe values (>7) found in benign cells. The difference in the pHe values of cancer and benign cells has the potential to turn into a tool to develop chemical processes that will act against cancer cell proliferation selectively with a reduced impact in benign cells. We hypothesize that CaS nanostructures can selectively dissociate into Ca2+ ions and sulfides in the acidic extracellular fluid of cancer cells but not in the basic microenvironment of benign cells. We have studied the reactions of CaS as a function of pH to simulate the different extracellular environments of cancer and benign cells using the Gibbs minimization approach in the Python environment. We found that the CaS readily dissociated in the acidic pH values to Ca2+ ions and sulfides. The sulfides are distributed in pH sensitive species that include gaseous H2S, aqueous H2S, HS‐ and S2‐ ions. The results are consistent with experimental observations of H2S production from CaS as a function of pH performed in the laboratory bench. The results are discussed in the context of possible chemical process that take advantage of the acidic microenvironment in cancer tissues to selectively induce apoptosis in the cancer cells with no effect in the corresponding benign cells, reducing secondary effects associated with chemotherapies in the market today.
Activation of Sympathetic Nervous System Contributes to Erthroprotein Gene Upregulation by Hypobaric Hypoxia
Authors: Su Xiaoyu , Matthews Timothy , Hildreth Matthew , Peng Ying‐Jie , Nanduri Jayasri , Prabhakar Nanduri R.
Published: May 2022

Hypobaric hypoxia (HH) experienced at high altitude initiates a series of physiological adaptations to maintain homeostasis. One such adaptation is the activation of the Epo gene which encodes the glycoprotein hormone erythropoietin (EPO). Increased circulating EPO levels defend against HH by facilitating blood O2 transport through increasing red blood cell production. HH also increases blood pressure (BP) through activation of the sympathetic nervous system and the ensuing elevation of circulating catecholamine levels. The present study examined the role of sympathetic nerve activation in HH‐evoked Epo gene activation and elevated circulating levels of EPO. Studies were performed on adult, male wild‐type (WT) mice treated with 18 hours of HH (0.4 atmospheres), simulating low O2 condition experienced at high altitude. Our results showed increased renal Epo gene abundance and elevated plasma EPO levels. Remarkably, these responses were either markedly attenuated or absent in mice with selective ablation of adrenal medulla as well as after treatment with propranolol, a non‐selective beta‐adrenergic receptor blocker. Moreover, infusion of epinephrine alone activated renal Epo gene and elevated plasma EPO levels. These results suggest that sympathetic nerve activation and the ensuing increase in plasma epinephrine contribute to Epo gene activation by HH.