bluuebunny/crossref_metadata_2025 huggingface dataset:
Females Have Altered Hemodynamics during Exercise Recovery with Caffeine Consumption
Authors: Grigoriadis Georgios , Bunsawat Kanokwan , Hilgenkamp Thessa Irena , Baynard Tracy
Published: November 2023

BACKGROUND

Caffeine consumption alters blood pressure (BP) and heart rate (HR) via sympathetic stimulation and arterial stiffness, as well as smooth muscle contraction. HR and BP recovery from a stimulus, such as exercise, can provide insight into aspects of autonomic control. Sex differences have been reported for HR recovery (HRR) and BP recovery (BPR); however, it is unclear if sex differences in recovery would still exist under an exacerbated adrenergic condition (e.g. caffeine).

PURPOSE

To examine sex differences in BPR and HRR with caffeine consumption following maximal exercise.

METHODS

Ten male (25 ± 3 yrs; 25.3 ± 3.5 kg/m2) and 8 female (26 ± 3 yrs; 22.8 + 2.4 kg/m2) individuals participated in a randomized, crossover study, with 400 mg of caffeine or placebo capsules ingested on 2 separate days. Each visit consisted of a maximal treadmill exercise. Beat‐to‐beat BP collected at rest, 5 and 15 min into passive recovery using finger photoplethysmography. HRR was recorded at the standard 1 and 2 min post‐exercise. Systolic (SAP), diastolic (DAP), mean (MAP), and pulse pressure (PP) change values from baseline rest to 5 and 15 min post‐exercise are reported in the table.

RESULTS

HRR, DAP, and MAP were not different between sexes for either placebo or caffeine. A sex x condition interaction (p<0.05) for the change in SAP and PP from baseline rest to 15 min was found. At 15 min post‐exercise, females had an increase in PP (p<0.001), whereas PP decreased in males.

CONCLUSION

Caffeine consumption did yield sex differences in BPR, with females having higher SAP and PP during the recovery period from maximal exercise versus males. The difference in SAP and PP with caffeine may be due to delayed parasympathetic activity in females compared to males.

MALE
FEMALE

PLACEBO
CAFFEINE
PLACEBO
CAFFEINE

ΔHRR1min
26 ± 11
26 ± 11
28 ± 12
31 ± 14

ΔHRR2min
48 ± 14
47 ± 12
48 ± 18
47 ± 15

ΔSAP5min
5 ± 10
−6 ± 11
0.7 ± 8
3 ± 10

ΔSAP15min
5 ± 11
−6 ± 12
−1± 6
4 ± 10

ΔDAP5min
−2 ± 8
−9 ± 9
−5 ± 6
−5 ± 9

ΔDAP15min
−1 ± 8
−9 ± 9
−4 ± 4
−5 ± 7

ΔMAP5min
1 ± 8
−8 ± 9
−2± 7
−1± 11

ΔMAP15min
2 ± 9
−8 ± 9
−2 ± 5
−1 ± 9

ΔPP5min
6 ± 7
3 ± 7
6 ± 6
8 ± 6

ΔPP15min
7 ± 6
3 ± 8
3 ± 6
9 ± 5*

MEAN ± SD;

Significant different between placebo and caffeine (p<0.001)
“I Can Hear Heart Sounds Too, How About You?” Familiarizing First‐Graders to Heart Sounds With Homemade Stethoscopes and Encouraging Heart Healthy Eating Habits From an Early Age
Authors: Taylor Jessica C , Prior Barry , Porter Johnny
Published: November 2023

Purpose

The purpose of this outreach was to familiarize elementary students with the concept of heart sounds heard through rudimentary homemade stethoscopes, heart rate detected by radial pulse, and to present healthy eating habits to support heart health during PhUn week 2015.

Methods

The team coordinated with the three first‐grade teachers at Sacred Heart Catholic School in Hattiesburg, MS. There were approximately 65 children that participated in the event. Paper towel cores were collected prior to PhUn week 2015 and delivered to the school approximately two weeks before the event date. Students were placed into pairs and given instructions for listening to heart sounds with a paper towel core “stethoscope.” One member of each pair, the “patient,” jumped up and down for 20 counts. Upon ceasing to jump the other member of the pair, the “physician,” placed the paper towel core stethoscope on the patient's chest and listened for the classic lub‐dub heart sounds. The students were then asked to switch roles and repeat the exercise. Medical student volunteers also demonstrated using two fingers to palpate the radial pulse and guided the students to find their own pulses. Finally, students were asked to place foods into two categories to demonstrate the role of nutrition in heart health. Pre‐selected foods were placed on one of two boards containing the titles, “Eat these foods more often” and “Eat these foods less often.”

Results

Approximately, one‐third of the students engaged during PhUn week 2015 had previously participated in this activity one or two times. Observational data suggests that previous exposure to this exercise predisposes the children to greater success in 1) hearing the heart sounds, 2) finding the radial pulse more easily than their classmates and 3) to answering physiology‐related questions, even with a one‐year absence in exposure to the outreach. Additional observational data suggests that sharing nutritional information helps students to consider wise food choices at an earlier age. Upon completion of the activity all of the children were aware that the heart beats constantly, that the heart makes sounds when it beats and that it is important to exercise and eat healthy foods for heart health. The children could also demonstrate the approximate size of their hearts by showing a clenched fist to classmates.

Conclusions

PhUn week is and continues to be an important avenue for introducing young students to physiology and increasing their scientific knowledge base. Also, repeated outreach exposures may increase students’ retention of physiologic concepts despite separation of the events by more than 12 months.
Measuring Brain Wave Activity in High School Students through Biopac Science Lab
Authors: Pawlak Robert , Evans LeVisa , Andreasen Nicholas , Anthis Taylor , Gurovich Alvaro N
Published: November 2023

The American Physiology Society (APS) has made it a goal to increase the understanding of physiology and promote careers in physiology. One of the APS initiatives that engages students is Physiology Understanding Week (PhUn). In this year's project, we used the Biopac MP40 system to measure brain function across high schoolers at South Vermillion High School in Clinton, IN. The activity was divided into 2 different days reaching 47 high schoolers taking two advanced anatomy and physiology classes. During the first day, 6 Biopac MP40 were available to both classes and 4 students per system collected data measuring brain function during 4 different conditions. The 4 conditions tested for brain function were 1) resting with eyes closed, 2) resting with eyes open, 3) sing a song in their head with eyes closed, and 4) read and solve a riddle. The second day, students analyzed the collected data and presented their main findings. The goal was to quantify the change in brain function when the difficulty of tasks is increased. While one student was tested other students gave commands and recorded data for alpha, beta, and delta waves. This data was then taken to construct charts so that the students could analyze the data for themselves. This activity helped the students apply their knowledge of neuro‐physiology to draw conclusion while expanding their knowledge of the brain. This interactive method enabled students to be more involved in their studies while taking an interesting new approach to physiology: a ‘PhUn’ approach.

Support or Funding Information

Supported by IU AHEC 15/16 #PIU535 and APS PhUn week program
Teaching Reaction Time using Applied Technology in High School Students
Authors: Anthis Taylor N , Brown Carson , Cole Shelby N , Crecelius Arick , Pawlak Robert , Jarrett Steven , Miller Breena , Gurovich Alvaro N
Published: November 2023

This project was designed to educate High School students on the importance of understanding neuro muscular physiology. Forty‐seven students from 2 upper anatomy/biology classes at South Vermillion High School in Clinton, IN learned how proprioception and nerve conduction velocity influence reaction time. The primary information was presented to the students in a PowerPoint and then demonstrated using an applied technology system (Biopac MP40). The Biopac MP40 is integrated with a laptop computer, and is used to show data by reading electrical impulses released by muscles. The Biopac MP40 synchronized two signals to determine a student's reaction time. It uses an acoustic signal, via headphones and the electrical signal sent by the electrodes placed on the student's arm to determine their reaction time. Twelve 4‐student small groups collected data from themselves. One student was the subject and the other three controlled the Biopac MP40 and wrote down the collected data. Finally, during a second day of this activity, students were asked to analyze and present the collected data, especially focusing on the difference between sex and arm dominance. This interactive method enabled students to be more involved in their studies while taking an interesting new approach to physiology: a ‘PhUn’ approach.

Support or Funding Information

Supported by IU AHEC 15/16 #PIU535 and APS PhUn week program
7th Grade Students at Moanalua Middle School Have PhUn with Exercise Physiology
Authors: Ho Dao H. , Pierce Lisa M. , Washington Michael A. , Campbell Carmen E. , Murata Lee‐Ann M. , Hernandez Claudia A. , Criman Erik T. , Peebles Melina , Uyehara Catherine F.T.
Published: November 2023

Researchscientists from the Department of Clinical Investigation at Tripler ArmyMedical Center (Honolulu, HI), brought PhUn to the classroom of Ms. MelinaPeebles at Moanalua Middle School (Honolulu, HI). Twenty‐eight 7th grade students spent the morning learning about science as a career choice, the importance of exercise in maintaining good health, and how cells, muscles, and the cardiovascular system all play a role in exercise physiology. The students participated in four activities, each relating to muscle physiology: 1)identifying different organelles of a cell and their functions, 2) learning about static and rhythmic exercise, 3) learning how exercise, such as dancing to music, affects heart rate, and 4) understanding how the cardiovascular system works to deliver oxygen to muscles. The pace and variety of the activities kept the students attentive, and excited about learning the “ins and outs” of exercise physiology. Some examples of how we made the activities engaging and educational for the students include: using a model of the heart and circulatory system to explain oxygen delivery to muscles, using stethoscopes and cell phone apps to measure heart rate, and using “shrinky dink” film to allow students to make their own key chain of a cell. Overall, PhUn day at Moanalua Middle School was a great success.

Support or Funding Information

The views expressed in this abstract are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. Government.
A Day of Hands‐on Physiology Demonstrations Increases Understanding of and Excitement towards Physiology in a Native American Indian Population of Middle and High School Students
Authors: Becker Bryan K , Schiller Alicia M , Zucker Irving , Godfrey Maurice
Published: November 2023

Purpose

To provide middle and high school students from Native American Indian reservations from Nebraska and South Dakota with interactive physiology activities to increase their understanding and familiarity with physiology and excitement toward science.

Methods

An activity‐based “Physiology Day” was held for approximately 300 middle and high school students from various Native American Indian reservations from Nebraska and South Dakota. Students rotated through 9 stations in small groups to complete hands‐on activities over diverse areas of physiology such as respiration, digestion, cardiovascular control, special senses, and more. Prior to completing any of the activities, students were given a questionnaire to determine their baseline familiarity with and attitudes toward physiology and science. Following the activities, the students were given a second questionnaire to gauge both the effectiveness of the various demonstrations and the students’ self‐perception of their understanding of basic physiological concepts and excitement toward science.

Results

At the beginning of the day, 79% of students rightly identified Physiology as being the study of how the body works; however a large proportion of students (18%) identified physiology as the study of physics. Following the day of activities the proportion of students correctly defining physiology increased to 89%. When asked to evaluate the level of current understanding of physiology, only 1% of students identified as “knowing a lot” 64% identified as “knowing a little”, and over one third (35%) said they “know nothing” about physiology. Following the day of activities, students self‐evaluating that they “knew a lot” about physiology increased to 24%, “knowing a little” increased to 69% and “knowing nothing” dramatically decreased to 7%. All 9 of the activities ranked high in satisfaction and effectiveness with 82–94% of respondents saying they learned something or enjoyed the activity. We conclude that our hands‐on physiology demonstrations and activities are effective means by which to engage middle and high school students increasing both their knowledge and excitement toward physiology.

Support or Funding Information

SEPA (RR032178) (OD011071)
Re‐evaluation of the effect of the phosphatase inhibitor okadaic acid on aquaporin 2 phosphorylation and trafficking
Authors: Day Jack , Ueberdiek Lars , Cheung Pui W , Bouley Richard , Brown Dennis
Published: November 2023

Aquaporin‐2 (AQP2), the vasopressin (VP)‐sensitive water channel, plays an important role in water reabsorption. Accumulation of AQP2 on the plasma membrane in kidney collecting duct principal cells increases water permeability and is necessary for urine concentration. AQP2 trafficking is tightly regulated by phosphorylation and dephosphorylation, and the role of each of four phosphoserine residues (pS256, pS261, pS264 and pS269) has been intensively studied. Recent work in our laboratory has shown that erlotinib (Erl), an epidermal growth factor receptor (EGFR) inhibitor, induces AQP2 membrane accumulation in the absence of VP. Western blot analysis using selective phospho anti‐AQP2 antibodies showed a VP‐like effect. Here, we investigate how phosphatase inhibition affects trafficking and intracellular location of AQP2 in the presence of VP and erlotinib. We performed immunohistochemistry and western blotting on LLC‐PK1 cells stably expressing WT AQP2. Cells were treated with okadaic acid (OA, 1 μM), a phosphatase inhibitor specific for PP1 and PP2A, for 10 minutes before treatment with either VP (10 nM) or Erl (2 μM). Interestingly, OA treatment alone did not significantly increase AQP2 membrane accumulation in our cell model, in contrast to a previous report in CD 8 cells (Valenti et al 2000), but did modify its intracellular distribution into a perinuclear position. Despite the lack of AQP2 membrane accumulation, western blot analysis showed that OA alone increases phosphorylation at all four phosphorylation sites. The VP or Erl induced AQP2 membrane accumulation is not affected by OA, but unexpectedly, pretreatment with OA did not inhibit VP or Erl induced S261 dephosphorylation. This suggests that phosphoserines 256 and 269 are the main substrates of phosphatases inhibited by OA (i.e., PP1, PP2a) and that serine 261 is the substrate for an OA insensitive phosphatase activated by VP and Erl.

Support or Funding Information

The American Physiological Society supported this work
Effects of NBCn1 deletion on behavioral actions of ethanol in mice
Authors: Choi Inyeong , Jones Jayre , Chung Jaegwon , Lee Jae‐Kyung , Schank Jesse
Published: November 2023

Genomic studies have been directed towards screening the gene maps of a large number of drug abusers and non‐abusers for genetic differences that might explain the propensity towards addiction. Studies show that the human SLC4A7 gene has genome‐wise association with multidrug abusers in the US population. The SLC4A7 gene encodes the sodium‐bicarbonate cotransporter NBCn1, the membrane protein that moves HCO3− into the cytosol and keeps intracellular pH from falling below normal. In the central nervous system, NBCn1 is particularly important for maintaining pH at synapses, where a small pH change in the synaptic cleft exerts significant effects on membrane excitability and complex firing patterns. In this study, we examined the effect of NBCn1 deletion on behavioral actions of ethanol in mice. Slc4a7 knockout mice lack the functional NBCn1 and develop abnormal pH in neurons and these animals are useful to gain knowledge of pH effects on alcoholism features. Knockout mice and wildtype mice were injected with 3.5 g/kg ethanol and the loss of righting reflex was examined to determine alcohol sedation. Knockout mice showed a 33% increase in the sleep time relative to the wildtype. The sleep latency (onset after injection) was not significantly different between wildtypes and knockout mice. In two bottle choice drinking experiment, mice were given to 3–12% alcohol, 4 days at each concentration. The mutant mice had alcohol consumption by 50% more than the controls at every concentration. Thus, the NBCn1 knockout mice drink more alcohol and also show increased sensitivity to alcohol‐induced sedation. To examine the effects of alcohol on glutamate signaling in knockout mice, we performed extracellular recordings of the CA3 region in hippocampal slices and found decreased NMDA receptor activity in knockout mice. Knockout mice have low NMDA receptor expressions determined by immunoblots with NR1 and NR2 antibodies. In adult wild type mice that were chronically administered with ethanol (>1 month), NBCn1 mRNA was downregulated in the hippocampus and midbrain compared to untreated control mice determined by qPCR. These results are consistent with our hypothesis that pH regulated by NBCn1 contributes to the susceptibility to ethanol. The lack of NBCn1 in KO mice lowers cell pH and makes mice more susceptible to ethanol, and a similar effect might occur when NBCn1‐dependent pH regulation is altered by an abnormal NBCn1.

Support or Funding Information

American Heart Association; NIH; Emory URC grant
Defining the Mechanism of Erythropoietin's Vasoactive Properties in Resistance Arteries
Authors: Kilar Cody R , Diao YanPeng , Sautina Larysa , Mohandas Rajesh , Conrad Kirk P , Segal Mark S
Published: November 2023

Clinically, erythropoietin (EPO) is known to increase systemic vascular resistance, however, in cell culture, EPO has been reported to increase activation of endothelial nitric oxide synthase (eNOS). How EPO can activate eNOS, while causing vasoconstriction, is likely due to EPO's ability to activate multiple receptors, the homodimeric EPO receptor (EPOR2) and the heterodimeric EPOR/beta common receptor (βCR). The purpose of this study was to investigate the contribution of each receptor in endothelial‐dependent vasodilation. We hypothesized that the vasoconstrictive effects seen with EPO administration are mediated through the EPOR2. To study the effects of each receptor, we utilized EPO and the EPO peptide (EPOp), which solely acts on the heterodimeric EPOR/βCR. Adult 16‐week old male C57BL/6 mice were euthanized and first order mesenteric arteries were isolated for in vitro study. Endothelium‐dependent (acetylcholine) and ‐independent (sodium nitroprusside) vasodilator responses were evaluated with and without an acute EPO (50 mIU/mL) or EPOp (25 ng/mL) pretreatment. We found that acetylcholine, endothelium‐dependent, vasodilation was impaired in arteries pretreated with both EPO and EPOp as compared to controls. However, while the EPOp vasodilation was impaired by 60%, EPO pretreatment totally inhibited acetylcholine‐induced vasodilation. Importantly EPO, EPOp and control treated arteries were all able to vasodilate equally in response to sodium nitroprusside, an endothelium‐independent vasodilator. These findings suggest that acute activation of the homodimeric EPOR2 or the heterodimeric EPOR/βCR can both lead to an endothelial specific impairment of vasodilator function in resistance arteries. We will discuss the putative mechanism of these effects along with the physiologic significance.
Microfluidic Models of the Microvasculature for Red Blood Cell Metrology in Sickle Cell Disease
Authors: Betz Jordan , LaVan David , Ackerman Hans
Published: November 2023

The microvasculature plays an important role in the pathology of sickle cell disease. As erythrocytes deliver oxygen while traversing the arterial and capillary networks, deoxygenated sickle hemoglobin begins to polymerize into fibers, causing changes in erythrocyte mechanical and adhesive properties that contribute to the obstruction of capillaries and postcapillary venules. Micropipette aspiration, atomic force microscopy, optical tweezers, and other related experimental techniques have been used to measure mechanical and adhesive properties of erythrocytes, but these approaches require static conditions that ignore the effects of shear stress. More recently, microfluidic devices have been introduced that are capable of providing realistic shear rates, but employ channel diameters that are much larger than those of human capillaries. We used photolithography and soft lithography techniques to fabricate microfluidic devices from (poly)dimethylsiloxane that recapitulate the internal dimensions of human capillaries. The physical constraints imposed by these channels allow us to measure the dynamics of deformation and “reformation” ‐ how quickly a red cell recovers its biconcave disc shape upon exit from a capillary channel.

In our devices, the initial inlet channel bifurcates five times, resulting in 32 capillaries that are 4 μm wide and 1 mm long. The channels are 2.5 μm tall, which orients the erythrocytes flat and keeps them within the focal plane of a microscope objective lens. We used high speed phase contrast video microscopy to record and analyze the cells under physiologically representative flow conditions at1% hematocrit. We measured the differences in erythrocyte elongation (the ratio of the maximum and minimum Feret diameter) and circularity (4π*Area/Perimeter2), as well as characterizing the time to return from the constrained shape in the capillaries to the more relaxed discoid shape, in sickle cell patients and healthy controls under both oxygenated and deoxygenated conditions. These measurements provide quantitative assessment of erythrocyte deformability and shape recovery that we will use to study vascular occlusion that occurs in sickle cell disease.

Support or Funding Information

All samples were drawn from volunteers with informed consent under protocol 03‐H‐0015 as reviewed and approved by the National Heart, Lung, and Blood Institute's Institutional Review Board. This research was supported by the Division of Intramural Research of the National Heart, Lung, and Blood Institute and the Material Measurement Laboratory of the National Institute of Standards and Technology.
A Diet High in Fat and Cholesterol Reduces Flow‐induced Dilations of Small Visceral Arteries and Involves Kir Channel Impairment
Authors: Fancher Ibra S , Ahn Sang Joon , Bian Jing‐Tan , Phillips Shane A , Levitan Irena
Published: November 2023

Distinct mechanisms regarding the progression of vascular endothelial dysfunction in diet‐induced obesity remain largely unknown. A diet high in saturated fat and cholesterol promotes vascular dysfunction prior to more severe disease states. Vascular K+ channels have also been implicated in the progression of vascular disease with obesity and our group established a definitive effect of dyslipidemia on Kir channel function. In this study we investigated the role of flow‐induced Kir2 channel activation and suggest Kir2 channel impairment in 1) a mouse model of diet‐induced obesity and 2) morbidly obese humans. We expand our investigation to spatially distinct arteries isolated from visceral and subcutaneous adipose. We show that a high fat, high cholesterol diet reduces flow‐induced vasodialtion in visceral arteries of mice and humans while subcutaneous endothelium‐dependent function remains intact. Importantly, barium reduces flow‐induced vasodilations in normal fed mice and in subcutaneous arteries, but has no effect on visceral arteries of obese mice or humans, implicating spatially distinct Kir channel dysfunction in obesity. Interestingly, visceral endothelial‐dysfunction correlates with elevated serum LDL. Our future direction will focus on the molecular mechanisms of Kir channel dysfunction in diet‐induced obesity.

Support or Funding Information

5 T32 DK080674 and

NIH R01 HL073965
Lysophosphatidic Acid Promotes a Switch from Nitric Oxide to Hydrogen Peroxide as the Mediator of Flow‐Induced Dilation in Human Arterioles
Authors: Chabowski Dawid S. , Kadlec Andrew O. , Ait‐Aissa Karima , Hockenberry Joseph C. , Beyer Andreas M. , Gutterman David D.
Published: November 2023

Introduction

Microvascular dysfunction is strongly predictive of cardiovascular pathology. Previous studies in our laboratory have shown that flow‐induced dilation (FID) in patients with coronary artery disease (CAD) is dependent on hydrogen peroxide (H2O2) as opposed to the more vasculoprotective nitric oxide (NO)‐mediated mechanism observed in arterioles from patients without CAD. Lipid phosphate phosphatase 3 (LPP3) is an integral membrane protein that dephosphorylates lysophosphatidic acid (LPA). Large genome‐wide association studies have identified a single nucleotide polymorphism in PPAP2B, a gene encoding LPP3, as a predictor of CAD independent of traditional risk factors. A pro‐inflammatory endothelial phenotype with decreased levels of endothelial nitric oxide synthase (eNOS) and atherosclerosis occurs with reduced LPP3 expression and an increase in LPA. We hypothesize that treatment of healthy microvessels with LPA induces a switch in mediator of FID from NO to H2O2, recapitulating the disease state.

Methods

Human arterioles were isolated from healthy adipose and prepared for video‐ and fluorescence microscopy. LPA (10 μmol/L) was added to the bath for 30 minutes prior to constriction with Endothelin‐1. Internal diameters were measured at steady‐state during graded increases in intraluminal pressure gradients (flow) in the presence/absence of a NOS inhibitor (L‐NAME; 10−4 M) or H2O2 scavenger (PEG‐catalase; 500 units/mL). MitoPY1 fluorescent probe was used to detect mitochondrial H2O2 in response to flow in the presence and absence of LPA and PEG‐catalase.

Results

Acute exposure to LPA reduced FID compared to control vessels (% max dilation, LPA: 61.7±6.0, n=4 vs. Control: 77.7±3, n=2). PEG‐catalase largely suppressed the remaining dilation (LPA + PEG‐catalase: 17.2±6.8, n=3). Interestingly, treatment with L‐NAME improved FID compared to LPA alone (LPA+LNAME: 83.7±6.8, n=3 vs. LPA: 61.6±6.0, n=4). LPA increased flow‐induced mitochondrial H2O2 production (% increase to flow, Control: −1±25, n=3 vs. LPA: 154±40, n=4). PEG‐catalase suppressed the increase in LPA‐induced, flow‐stimulated H2O2 production (PEG‐cat: 37±19, n=2). In summary, LPA is capable of shifting the mediator of dilation from NO to H2O2 in healthy arterioles. In addition, in the presence of LPA, blocking NOS paradoxically improves FID, raising the question whether NOS is uncoupled in this situation. Mitochondria appear to be the source of LPA‐induced increase in H2O2. We conclude that LPA may play a role in microvascular dysfunction in CAD, invoking changes in NOS and ROS formation.
The microtubule‐associated End Binding proteins regulate the ER exporting and trafficking of the TRPM4 channels
Authors: Romero Anibal , Blanco Constanza , Morales Danna , Mogollones Ignacio , Álvarez Alhejandra , Varela Diego , Cáceres Mónica , Cerda Oscar
Published: November 2023

Trafficking and localization of ion channels are critical events for their proper function. These processes are relevant in several cellular functions, such as developmental programmes, cell polarity and local membrane potential. TRPM4 is a Ca2+‐activated non‐selective cationic channel expressed in different tissues and cell types, and is involved in a wide variety of physiological processes, such as insulin secretion and mechanotransduction. Altered activity, expression and localization of these channels might trigger several pathophysiological conditions. Indeed, TRPM4 plasma membrane overexpression has been related to cardiovascular and neurodegenerative diseases and cancer. Therefore, the mechanisms involved in the regulation of TRPM4 activity constitute an important area of biomedical research. Protein‐Protein Interactions (PPIs) control the expression, trafficking, localization and biophysical properties of ion channels. Thus, the identification of novel TRPM4‐related PPIs and their characterization might contribute to dissect the regulatory mechanisms of this channel. Interestingly, bioinformatics analyses of the primary sequence of TRPM4 allowed us to identify a putative interacting motif to End Binding (EB) proteins, novel members of the microtubule plus‐end tracking proteins (+TIPs). These proteins bind a consensus motif (SxIP) in their substrates and are involved in a plethora of cellular processes, including growing dynamics of the microtubule cytoskeleton, focal adhesion dynamics, cell migration and protein trafficking, targeting and localization. Here, we provide novel data suggesting that TRPM4 interacts with EB proteins. Moreover, we show that the mutation (TRPM4ΔSWIP and TRPM4SWNN) of the putative EB‐binding motif abolishes the TRPM4‐EB interaction. We also found that these mutant variants show a reduced expression of the mature population of the channel and display an endoplasmic reticulum (ER)‐like distribution. Furthermore, these mutant variants present a decreased expression in the plasma membrane, consistent with a reduction in the whole‐cell electrophysiological recordings. Conversely, we demonstrated that the expression of a soluble fragment containing the wild type N‐terminal region of TRPM4 (EGFP‐N‐TRPM4WT) reduces the membrane expression of TRPM4. These results suggest that EB interaction is necessary for the proper trafficking and activity of TRPM4. These findings might contribute to the understanding and characterization of novel mechanisms involved in TRP channel trafficking/localization, and could be applied to the design of therapeutic strategies against TRPM4 gain‐of‐function‐associated pathologies.

Support or Funding Information

This research was funded by Fondecyt 11121239 to O.C. and Fondecyt 11140064 to M.C. A.R. is a Conicyt Doctoral Fellowship recipient.
Salidroside Inhibits Clinorotation‐induced Apoptosis in Pulmonary Microvascular Endothelial Cells by regulating signaling pathway of Endoplasmic reticulum stress
Authors: Kang Chunyan , YUAN Ming , WANG Jingyu , YUAN Min , LIU Changting
Published: November 2023

Objective

Microgravity or simulated microgravity induces dysfunction of endothelium. We have previously demonstrated that clinorotation induces apoptosis in microvascular endothelial cells. Salidroside(Sal), one of main active ingredients extracted from the root of Rhodiola rosea, has been documented to have potent antiapoptotic effects on some cells. The present study investigated whether salidroside inhibited clinorotation‐ induced apoptosis in microvascular endothelial cells by regulating the signaling pathway of endoplasmic reticulum(ER) stress.

Methods

Human pulmonary microvascular endothelial cells(HPMECs) were divided into control group, clinorotation group, clinorotation+Sal and clinorotation+Sal+ LY294002 pretreatment groups. Microgravity was simulated by clinorotation. After 72h clinorotation, the apoptotic rate of HPMECs was detected by TUENL assay and JC‐1 staining. The protein expressions of PI3K, Akt, Bcl‐2, Bax, caspase‐3 and caspase‐9 were determined by Western blot. Meanwhile, the ER stress related proteins such as GRP78, CHOP, IRE1a and ATF6 were measured.

Results

TUNEL assay, together with JC‐1 staining indicated that 72h clinorotation significantly induced apoptosis in HPMECs, which was reversed by salidroside treatment. Clinorotation also decreased the protein expression of PI3K, Bcl‐2 and phospho‐Akt level, increased the apoptosis related protein expression of Bax, caspase 3 and caspase 9. Meanwhile, Clinorotation increased the ER sensors protein expression of GRP78, CHOP, IRE1a and ATF6. Pretreatment with salidroside reversed the changes of these protein expression induced by 72h clinorotation, however, the protective effects of salidroside were reversed by inhibitor of PI3K/Akt, LY294002.

Conclusion

Our results suggested that the apoptosis of HPMECs induced by 72h clinorotation can be alleviated by pretreatment of salidroside. The protective effects of salidroside on HPMECs apoptosis may come from the upregulation of PI3K/Akt signal pathway and downregulation of ER stress pathway.

Support or Funding Information

Funded by the Defense Medical Fund of China (Grant No. 06Z048) and State key lab of space medicine fundamentals and application, China Astronaut Research and Training Center (grant No. SMFA14B01).
Lipid Raft Disruption Selectively Blocks the Cell‐Signaling Activity of LRP1 in Neuron‐Like Cells
Authors: Laudati Emilia , Gilder Andrew S. , Lam Michael S. , Gonias Steven L. , Mantuano Elisabetta
Published: November 2023

LDL receptor‐related protein‐1 (LRP1) is an endocytic and phagocytic receptor. In neurons and neuron‐like cells, binding and endocytosis of specific ligands is coupled to robust cell‐signaling responses. The majority of the LRP1, present at the cell‐surface in most cells, localizes to clathrin‐coated pits. However, in some cells, LRP1 also local‐izes to lipid rafts. We previously demonstrated that, in PC12 and N2A neuron‐like cells, tissue‐type plasminogen activator (tPA) and activated α2‐macroglobulin (α2M*) phos‐phorylate TrkA and activate ERK1/2 in a response that requires LRP1 and the NMDA Receptor. We have now shown that in PC12 and N2A cells, LRP1 is partially localized to lipid rafts. In these cells, both enzymatically‐inactive tPA and α2M* acti‐vated ERK1/2, confirming our earlier result. When PC12 or N2A cells were treated with methyl‐β‐cyclodextrin (MβCD), to disrupt lipid rafts, cell‐signaling in response to tPA and α2M* was blocked. MβCD had no effect on the total cellular abundance of LRP1, as deter‐mined by immunoblot analysis or on the cell‐surface abundance of LRP1, as deter‐mined by measuring the binding of radioiodinated α2M* at 4º C. MβCD also failed to af‐fect endocytosis of radioiodinated α2M* at 37º C. The selective effects of MβCD on LRP1‐initiated cell‐signaling was apparent in neuritogenesis experiments in PC12 cells; when lipid rafts were disrupted, tPA and α2M* failed to induce neurite outgrowth, which was observed in control cultures. Our studies suggest that LRP1 signaling is initiated in lipid rafts. The fact that LRP1 localizes to lipid rafts only in some cell types may partially explain why its activity in cell‐signaling is cell type‐specific.
Tolfenamic acid downregulates β‐catenin in colon cancer
Authors: Lee Seong‐Ho , Ha Taekyu , Lou Zhiyuan
Published: November 2023

Tolfenamic acid, a nonsteroidal anti‐inflammatory drug (NSAIDs) has been shown to induce apoptosis and suppress cancer cell growth and tumorigenesis in colon cancer. Wnt/β‐catenin pathway is highly activated in colon cancer and considered as target for cancer prevention and therapy. The current study was performed to study if tolfenamic acid affects β‐catenin pathway in colon cancer. Tolfenamic acid resulted in an inhibition of cell growth and downregulation of β‐catenin in dose‐ and time‐dependent manner in different types of human colon cancer cells. Reduction of β‐catenin was associated with ubiquitin‐mediated proteasomal degradation. Treatment of tolfenamic acid to colon cancer cells downregulated expression of Smad2 and Smad3. Overexpression of Smad2, but not Smad3 blocked tolfenamic acid‐induced downregulation of β‐catenin. Decreased expression of β‐catenin, Smad2, Smad3 and VEGF were observed in tumor tissues of mice treated with tolfenamic acid at dose of 50 mg/kg body weight. Our findings suggest that tolfenamic acid inhibited cancer cell growth by inducing β‐catenin degradation via ubiquitin‐proteasome pathway and Smad2 mediated tolfenamic acid‐induced downregulation of β‐catenin.

Support or Funding Information

American Cancer Society grant (RSG‐11‐133‐01‐CCE) to S‐H Lee
Paks integrate polarity and vesicle transport signaling in ovarian epithelial cancers
Authors: Radu Maria , Mumber Hannah , Chernoff Jonathan
Published: November 2023

P21 activated kinases (Paks) are effectors for small RhoGTPases and have been widely implicated in regulation of cytoskeleton arrangements and cell architecture. Paks gained a lot of momentum when several members of the family have been shown to be over‐expressed in a variety of cancers, including ovarian, breast, prostate and colon. These discoveries led to augmented efforts to discover new and improved Pak inhibitors to be used in clinic. Recent data shows however that the absence or complete inhibition of Pak2 leads to lethality in adult animals. It is thus imperative that we have a better understanding of Pak regulated pathways and their direct substrates.

In an effort to unveil new molecular efforts in Pak signaling, we employed a modified kinase assay in conjuncture with peptide identification by LC/MS. A high percentage of the newly discovered Pak substrates fell in the category of molecules that control cell communication. Cell communication relates to all modes of inter and intra cell messaging, including cytoskeleton arrangement and vesicle transport. Importantly, these are also key elements that when deregulated lead to epithelial cancer initiation and progression.

Among the newly identified Pak direct targets that regulate cell communication CDK16 and NSF have been previously shown to regulate each other and together to control polarity and vesicle transport respectively. We found that Pak1 and Pak2 phosphorylate CDK16 at S119 and NSF at S207. Our data has shown that unphosphorylatable CDK16 mutants are unable to bind Pak1 and 2 and that inactive Paks do not bind CDK16. This suggests that functional protein complexes are formed between CDK16 and Pak1 and 2. Furthermore, we found that in an ovarian carcinoma cell line overexpression of an unphosphorylatable CDK16 mutants can induce stress fiber and focal adhesion disassembly (link to deregulated polarity), while a CDK16 mutant that mimics a constitutively phosphorylated state can partially rescue stress fiber disassembly induced by Pak inhibition.

These data strengthen our hypothesis that Paks regulate epithelial cancer progression by phosphorylating CDK16 and NSF, proteins that integrate polarity and vesicle trafficking pathways. To confirm this hypothesis we will first investigate the temporal and spatial regulatory events that take place between Pak, CDK16 and NSF and how these events influence polarity and vesicle transport. Secondly, we will investigate how the dis regulation of Pak/CDK16/NSF pathway leads to epithelial cancer initiation and spread. Thirdly, we will consider the possibility of combining Pak and CDK16 inhibition to arrest the growth various epithelial carcinomas.

Our results address the possibility to target Pak signaling in Pak overexpressing cancers by inhibiting CDK16, a downstream kinase effector and to override some of the critical effects of current Pak inhibitors.

Support or Funding Information

NIH T32 Training grant; NCI CA06927 to Fox Chase Cancer Center
Association between Polymorphism of Genes Encoding for 5‐HT1A and 5‐HT2A Receptors and Response to Selective Serotonin Reuptake Inhibitors in Malaysian Patients with Major Depressive Disorder
Authors: Badamasi Ibrahim Mohammed , Lye Munn Sann , Ibrahim Normala , Ling King Hwa , Stanslas Johnson
Published: November 2023

Inter‐racial variations in genotype frequency have been implicated as a probable cause of the inconsistencies in pharmacogenetic studies of major depressive disorder (MDD). The aim of this study was to determine whether the polymorphisms of the serotonin 1A (5HT1A) 1019C>G and serotonin 2A (5HT2A) 1438A>G are associated with response to SSRI treatment among Malaysian MDD patients. These polymorphic changes are shown to modulate the transcription of these genes and are associated with attenuation of the density of the encoded receptors. Modulation of these receptors is central to the activity of antidepressant medication. The study involved six weeks follow up of incidence and prevalence (diagnosed within 2 years to recruitment) MDD patients from a case‐control hospital based study in Malaysia. Only Malaysians aged 18–65 years and diagnosed with MDD (other Axis 1 psychiatric diagnosis were excluded) on treatment with SSRIs were recruited. One hundred and ninety two patients (females ‐ 71.4% and males – 28.6%) were recruited. A total of 77.7% of the patient showed response to treatment and the rest did not. The response rate was also highest among the females (81%) compared to males (67%). The frequencies of the genotypes 5HT1A1019C>G were GC (34.2%), CC (55.2%), GG (10%) and those of the 5HT2A 1438A>G were GA (45.3%), AA (33%), GG (21.9%). Association testing for response to SSRIs with the 5HT1A 1019C>G polymorphism and not the 5HT2A1438A>G polymorphism revealed a chi‐square score of 3.911 with P≤0.05 (P=0.048) in a dominant model of inheritance. This study revealed that the dominant effect of the G‐allele in both the GG and GC genotypes of the 5HT1A receptor gene was associated with treatment response in Malaysian MDD patients.
Regulation of the yeast mitochondrial respiratory complex IV activity by the major isoform of the ADP‐ATP carrier
Authors: Ogunbona Oluwaseun Basit , Baile Matthew G , Claypool Steven M
Published: November 2023

Protein‐protein interactions underlie many biological processes. The ADP/ATP carrier (AAC) forms an evolutionarily‐conserved, cardiolipin‐dependent interaction with the respiratory supercomplexes. Interestingly, in yeast lacking Aac2p the activity of respiratory complex IV (cytochrome c oxidase or COX) is reduced suggesting that this interaction is of functional significance. The regulation of COX activity and by extension, of oxidative phosphorylation by AACs is not clearly understood. Here, we leveraged a transport‐dead pathogenic AAC2 point mutant discovered in a patient with hypertrophic cardiomyopathy and mild myopathy to determine whether the reduction in complex IV activity in the absence of Aac2p reflects the absence of the interaction between AACs and components of the electron transport chain and/or the absence of nucleotide transport (i.e. Aac2p function). Using a CRISPR‐Cas9 based strategy, we established yeast strains with either an AAC2 frameshift deletion or an AAC2A137D point mutation in the presence or absence of the minor AAC isoforms, Aac1p and Aac3p. Importantly, the pathogenic A137D allele of AAC2 is expressed normally and still interacts with components of the yeast respiratory supercomplex but is unable to support growth on respiratory media due to its inability to transport ADP/ATP. Interestingly, complex IV activity is significantly reduced in yeast strains lacking Aac2p activity (P<0.05). Moreover, in the absence of Aac2p function, the expression levels of complex IV subunits encoded by the mitochondrial genome are specifically reduced. Thus these new pieces of evidence suggest that Aac2p nucleotide transport function and not the physical interaction with the respiratory supercomplexes is required for full COX activity. Mechanistically, the absence of Aac2 function specifically impairs expression of mitochondria DNA‐encoded COX subunits. Given that oxidative phosphorylation accounts for the majority of cellular ATP production, uncovering this important line of regulation is critical for further understanding of the various role the mitochondria play in healthy and diseased state.

Support or Funding Information

“THIS WORK WAS SUPPORTED BY AMERICAN HEART ASSOCIATION GRANT #15PRE24480066 (Oluwaseun Ogunbona) and NATIONAL INSTITUTES OF HEALTH GRANT NIH/NHLBI 1R01HL108882 ‐ 01A1 (Steven Claypool)
Acceleration of Skin Barrier Restoration via Activation of Transient Receptor Potential Vanilloid 3 (TRPV3) by Agrimonia pilosa Leaf
Authors: Nam Joohyun , Kim Woo Kyung
Published: November 2023

The formation and maintenance of the skin barrier are controlled by the proliferation and differentiation of epidermal keratinocytes, and impaired skin barrier homeostasis resulting from abnormal keratinocyte function causes many skin disorders, including atopic dermatitis and psoriasis. Keratinocyte differentiation is regulated by intra‐ and extracellular calcium ion concentrations. Recently, it was reported that the transient receptor potential vanilloid 3 (TRPV3) ion channel is functionally expressed in human keratinocytes. Activation of TRPV3 increased intracellular calcium signaling, which in turn increased transglutaminase 1, 3 activities as well as the subsequent formation of cornified cell envelopes in the epidermis. It was also reported that activation of TRPV3 promotes epithelial cell proliferation and wound healing in oral epithelia. Thus, TRPV3 activation may be a potential therapeutic target for skin barrier recovery in various dermatological diseases. The leaves of Agrimonia pilosa Ledeb (Rosaceae, AP), which have traditionally been used in East Asia to treat conditions including sore throat, parasitic infection, and eczema, have been reported to possess anti‐inflammatory and anti‐allergic effects in lipopolysaccharide‐ and ovalbumin‐induced mouse models. We performed whole‐cell patch‐clamp and tape stripping tests to determine whether AP extract enhances barrier recovery via TRPV3 activation. We found that APH2O potently activated human TRPV3 and subsequently activated TGase activity in vitro. We also confirmed that topical application of APH2O improved the recovery of skin barrier disruption induced by tape stripping in mice. APH2O may be useful for preventing and treating skin barrier disorders, including inflammatory skin diseases such as atopic dermatitis and psoriasis.

Support or Funding Information

This research was supported by the Convergence of Conventional Medicine and Traditional Koran Medicine R&D program funded by the Ministry of Health & Welfare through the Korean Health Industry Development Institute (SHIDI) (To. JH Nam, HI15C0256).