bluuebunny/crossref_metadata_2025 huggingface dataset:
Celecoxib and Cardiac Abnormalities: a Biostatistical Analysis of Adverse Side Effects
Authors: Rusnak Kathleen , Roloff Greg , Singh Satpal
Published: June 2021

Celecoxib is a member of the coxib class of inhibitors that selectively inhibits COX‐2 over COX‐1. The withdrawal of rofecoxib (Vioxx) and valdecoxib (Bextra) due to severe cardiovascular complications has highlighted the importance of both pharmacological and epidemiological post‐market surveillance of this class of drugs. Previous work in our laboratory has shown that celecoxib can cause arrhythmic Drosophila heartbeat and the arrhythmic beating of rat heart cells in culture. This occurs via a mechanism that is independent of COX‐2 inhibition. Also, celecoxib inhibits the delayed rectifier potassium channel (KV2) in Drosophila and in rat heart cells, and the hERG potassium channels expressed in HEK‐293 cells. Since drug inhibition of hERG channels leads to cardiac arrhythmia in humans, it is important to examine clinical implications of the inhibition of these channels by celecoxib. We reviewed quarterly reports of celecoxib‐associated adverse events submitted to the United States Food and Drug Administration's (FDA) Adverse Events Reporting System over an eight‐year period. Data mining algorithms were developed to ascertain the number of celecoxib users and non‐users and the number of individuals in each group experiencing dysfunction of cardiac rhythm. We found that there was a statistically significant increase (unpaired t‐test p < 0.00001) of reported adverse events for those taking celecoxib (N= 48,824; 6.250%) compared to those not taking the drug (N= 3,600,644; 4.281%).
Molecular basis of pH sensitivity of Orai1/stim1 channels
Authors: Yue Zhichao , Tsujikawa Hiroto , Xie Jia , Yang Wenzhong , Yue Lixia
Published: June 2021

Changes of intracellular and extracellular pH are involved in a variety of physiological and pathological processes, in which regulation of the Ca2+ release activated Ca2+ channel (ICRAC) by pH has been implicated. Ca2+ entry mediated by ICRAC as well as native ICRAC currents have been shown to be regulated by acidic or alkaline pH. However, the mechanism by which the CRAC channel is regulated by internal pH (pHi) and external pH (pHo) has remained elusive. By generating a series of mutations in the channel pore region, intracellular and extracellular loops, transmembrane domains, and N‐ and C‐termini, we unexpectedly identified that the mutant E190D lost sensitivity to both acidic and alkaline pHo, and the mutant H155F markedly diminished the response to acidic and alkaline pHi. Thus, it appears that E190 in the TM3 is the external pH sensor, and H155 in the intracellular loop is the internal pHi sensor of Orai1/Stim1 channels. Our results provide a novel mechanism that changes of external and internal pH modulate ICRAC activity by directly regulating channel gating of the pore‐forming subunit Orai through residues E190 and H155. Given that pH changes can influence a variety of physiological/pathological functions, Orai/Stim channels may be an important mediator for various physiological and pathological processes associated with acidosis and alkalinization.
Targeting the transient receptor potential‐melastatin‐like 7 (Trpm7) kinase domain with the first inhibitor, inhibited breast cancer cell migration, invasion and tumor metastasis.
Authors: Kaoud Tamer , Xie Xuemei , Park Jihyun , Tavares Clint , Mitra Shreya , Cano Micael , Tseng Chun‐Chia , Radwan Mohamed , Bartholomeusz Chandra , Dalby Kevin
Published: June 2021

TRPM7 (transient receptor potential melastatin 7) is a non‐selective cation channel fused to protein kinase domain at the C‐terminal whose activity is linked to the control of actomyosin contractility. TRPM7 mediates adhesion and migration of breast cancer cells and promotes breast tumor metastasis. The lack of cell‐permeable inhibitors of the kinase domain represents a barrier to understand the kinase function. Herein, we discovered the first small molecule (KD‐1) that targets TRPM7 kinase activity. Mg2+ starvation, which promotes TRPM7 kinase activity, induces phosphorylation of eEF2. Treatment of Mg2+‐starved HEK293 cells with KD‐1 decreased eEF2 phosphorylation, consistent with TRPM7 kinase activity suppression in‐cells. KD‐1 decreased the binding of Myosin IIB to TRPM7 in HEK293 and MDA‐MB‐231 cells. And when MDA‐MB‐231 cells were treated with increasing doses of KD‐1, no change in cell viability was seen. Interestingly, KD‐1 inhibited MDA‐MB‐231 cells migration and invasion that have been reported to be regulated by TRPM7 kinase activity. Finally, the bioluminescent signals (to assess metastasis) were significantly lower in KD‐1‐treated mice (25 and 50 mg/kg/day) than in mice treated with vehicle control (P 蠄 0.05, 2‐sided t‐test.). As a conclusion, inhibition of TRPM7 kinase activity can be a valid approach to reduce or block breast tumor progression and/or metastasis.
Identification of Polysulfonated Inhibitors that Target Dual Specificity Phosphatase 5 and Provide New Insights into the Binding Requirements for Dual‐Phosphate Substrate Pockets
Authors: Bongard Robert , Neumann Terrence , Span Elise , Kalous Kelsey , Gastonguay Adam , Kutty Raman , Nayak Jaladhi , Lepley Michael , Bohl Chris , Lange Rachel , Sarker Majher , Talipov Marat , Rathore Rajendra , Ramchandran Ramani , Sem Daniel
Published: June 2021

Dual specific phosphatase‐5 (DUSP5) plays a central role in vascular development and disease. We present a p‐nitrophenol phosphate (pNPP) based enzymatic assay to screen for inhibitors of the phosphatase domain of DUSP5. pNPP is a mimic of the phosphorylated tyrosine on the ERK2 substrate (pERK2) and binds with a Km of 7.6 + 0.5 mM. Docking followed by inhibitor verification using the pNPP assay identified a series of polysulfonated aromatic inhibitors that occupy the DUSP5 active site in the region that is likely occupied by the dual‐phosphorylated ERK2 substrate (pThr‐Glu‐pTyr). A first lead molecule has a naphthalene trisulfonate (NTS) core. A search for similar compounds in a drug database identified an analog that is FDA approved (making it a candidate for repurposing), and is a potent and competitive inhibitor (25 + 5 µM). Further screening and ligand optimization identified additional analogs, including two new scaffolds – one based on a diphenyl‐ether core, and another based on a fused tricyclic core; the latter formerly FDA approved as a drug for topical use. Modeling suggests these inhibitors may have broader relevance for mimicking the dual‐phosphate loops recognized by DUSPs, as they position sulfonates or other negatively charged groups where the two phosphates on the substrate peptide (pThr‐Glu‐pTyr) bind.
Perforating Corneal Injury in Rat and Pentadecapeptide BPC 157
Authors: Masnec Sanja , Kokot Antonio , Zlatar Mirna , Kalauz Miro , Radic Bozo , Klicek Robert , Drmic Domagoj , Lazic Ratimir , Seiwerth Sven , Sikiric Predrag
Published: June 2021

Based on its healing effects in various tissues, we hypothesized that the stable gastric pentadecapeptide BPC 157 heals corneal ulcerations in rats and effects corneal transparency.

A penetrant linear 2‐mm incision was made in the paralimbal region of the left cornea at the 5 o'clock position with a 20‐gauge MVR incision knife (Bausch & Lomb) at 45° under an operating microscope. Medication, comprising BPC 157 that was dissolved in distilled water at 2 pg/mL, 2 ng/mL, 2 µg/mL, 2 eye drops in the left eye per rat, was begun immediately after induction of the injury and continued every 8 h up to 120 h; controls received an equal volume of distilled water. In contrast to the poor healing response in controls, BPC 157 significantly accelerated the healing process in 2 µg and 2 ng BPC 157‐treated eyes, starting 24 h after the injury, and the fluorescein and Seidel tests became negative. The epithelial defects were completely healed at 72 h after the injury in the 2 µg BPC 157‐treated group and at 96 h after injury in the 2 ng BPC 157‐treated group. Aqueous cells were absent at 96 h and 120 h after injury in either 2 µg or 2 ng BPC 157‐treated groups, respectively.

In conclusion, BPC 157 effects the rapid regaining of corneal transparency. Whereas controls developed new vessels that grew from the limbus to the penetrated area, BPC 157‐treated rats generally had no new vessels, and those that did form in the limbus did not make contact with the penetrated area. Thus, BPC 157 eye drops successfully close perforating corneal incisions in rats. Supported by Grant 108‐1083570‐3635, Croatia
METABOLISM OF SEROTONIN IN MOUSE AND HUMAN HEART
Authors: Neumann Joachim , Jung Franziska , Gergs Ulrich , Hofmann Britt , Simm Andreas , Silber Rolf‐Edgar
Published: June 2021

Serotonin (5‐HT) can exert cardiac effects like increase in force of contraction and increase in beating rate. It is generally assumed that 5‐HT is generated in non‐cardiac tissue and reaches the heart via the blood. However, we hypothesized that 5‐HT might be produced within the heart. Hence, we studied the effect of the direct precursor of 5‐HT, that is 5‐hydroxytryptophane. Indeed, 5‐hydroxytryptophane (1‐100 µM) exerted a concentration dependent increase in force of contraction and in beating rate in isolated left and right atrial preparations of transgenic mice overexpressing the human 5‐HT4‐receptor. But 5‐hydroxytryptophane was 30 to 100 fold less potent than 5‐HT. The effect could be blocked by pre‐incubation with an inhibitor (100 µM NSD 1015) of the pacemaker enzyme for formation of 5‐HT, that is aromatic L‐amino acid decarboxylase. Moreover, in isolated electrically driven (1 Hz) trabeculae (n=10) from five patients (undergoing bypass surgery), a positive inotropic effect to 100 µM 5‐hydroxytryptophane was noted which could be completely abolished by a 5‐HT4‐receptor antagonist (GR113808). These data combined argue for the possibility that intracardiac formation of serotonin can occur and might be relevant in health and disease.
Insulin Attenuates Cardiac Myocyte Contractility via NADPH Oxidase: Implications for Diabetic Cardiomyopathy
Authors: Sartoretto Juliano , Steinhorn Benjamin , Romero Natalia , Kalwa Hermann , Michel Thomas
Published: June 2021

Insulin is known to modulate metabolic responses in cardiac myocytes (CM), yet the role of insulin in regulation of CM contractility is less well understood. Type 2 diabetes mellitus (T2D) leads to diabetic cardiomyopathy, yet little is known about the role of insulin in CM function. Here we explore the role of insulin on CM contractility in adult mouse CM. Insulin had no effect on basal CM contractility, but inhibited the increase in contractility seen after stimulation of ß‐adrenergic receptors (ß‐AR) by isoproterenol (ISO; 40+/‐5% inhibition, p<0.01, n=115‐124 cells). The attenuation of ISO‐stimulated contractility by insulin was blocked by the NADPH oxidase inhibitor apocynin, implicating reactive oxygen species (ROS) in cardiac insulin responses. We found that insulin inhibited ß‐AR stimulation of CM Ca2+ transients (35+/‐4% inhibition, p<0.01, n=39‐41 cells); again, insulin inhibition of this ß‐AR response was blocked by apocynin. We analyzed CM contractile responses in CM isolated from mice fed a high‐fat diet, a model of T2D. We found that the insulin‐promoted decrease in contractility was blocked in CM isolated from diabetic mice. The phosphorylation of insulin‐dependent signaling proteins (insulin receptor, IRS‐1, Akt) was effectively abrogated in diabetic mice, consistent with myocyte insulin resistance. These data establish a central role for NADPH oxidase in insulin‐dependent physiological responses in CM, and show that these responses are deranged in mice with T2D. These observations suggest that alterations in ROS‐ and insulin‐dependent pathways play a role in the pathophysiology of diabetic cardiomyopathy. Research support: NIH and AHA.
Effects of Dynamin‐Related Protein‐1 (DRP‐1) Inhibition with MDIVI‐1 on Myocardial Injury, Mitochondrial Respiration and Stress‐Signalling in the Murine Heart
Authors: Wendt Lauren , See Hoe Louise , Sachaphibulkij Karishma , Du Toit Eugene , Peart Jason , Headrick John
Published: June 2021

Mitochondrial fission protein, dynamin‐related protein‐1 (DRP‐1), may be a key determinant of cardiac resistance to ischemia‐reperfusion (I‐R) and other stresses. We examined the impact of I‐R on myocardial expression of DRP‐1 (together with Akt and Erk1/2), mitochondrial respiration, and injury in I‐R hearts in 4mo male C57Bl/6 mice subject to DRP‐1 inhibition. Functional recoveries of Langendorff perfused hearts subjected to 25 min ischemia/45 min reperfusion were significantly improved by pre‐treatment with 1 µM MDIVI‐1. Cell death (LDH efflux) was only modified by a higher 5 µM concentration. Protective effects were not replicated by an alternate inhibitor dynasore hydrate (1 µM). Ischemic insult repressed mitochondrial O2 consumption during early reperfusion, with a ~50% fall in complex I activity that was effectively reversed by MDIVI‐1. Functional benefits were associated with MDIVI‐1 dependent reductions in mitochondrial DRP‐1 expression and cytosolic Erk1/2 phosphorylation during I‐R, while phosphorylation of cardiac Akt was significantly augmented by MDIVI‐1. A similar pattern was evident in parallel studies of H2O2 (400 µM) challenged H9c2 myoblasts, with MDIVI‐1 negating oxidative‐stress Erk1/2 phosphorylation whereas phosphoactivation of Akt was preserved. These data support a key role for DRP‐1 in repressing I‐R tolerance, mitochondrial function, and protective Akt phosphorylation (whilst also augmenting Erk1/2 activation). This role of DRP‐1 in control of stress‐resistance, mitochondrial function and survival‐kinase signalling supports the potential value of targeting the protein to protect ischemic myocardium.
Unacylated Ghrelin Protects Hearts of Mice Subjected to Myocardial Ischemia/Reperfusion
Authors: Huynh David , Bessi Valerie , Ménard Liliane , Noll Christophe , Carpentier Andre , Ong Huy , Marleau Sylvie
Published: June 2021

Objective

Ghrelin can be found in the circulation under two different forms: acylated ghrelin (AG) and unacylated ghrelin (UAG). UAG, while being the predominant ghrelin isoform in circulation, is devoid of endocrine activity. Despite its inability to activate the classical ghrelin receptor, some studies have shown that UAG possesses cardiotropic properties. Our aim is to investigate the cardioprotective effects of UAG against myocardial ischemia‐reperfusion (MI/R) injury.

Methods

C57BL/6 mice were pretreated twice a day for 14 days with either AG or UAG at 200 nmol/kg/day. Myocardial ischemia was induced by the ligature of left anterior descending coronary artery for 30 min, followed by reperfusion for 6 or 48 h.

Results

Infarct sizes were reduced by 68 and 60% (P<0.001) in AG‐ (n=6) and UAG‐treated mice (n=5), respectively. Unlike the UAG‐treated group, mice treated with AG showed increased fractional uptake rate (Ki) and net (Km) uptake of free fatty acids (FFA), by 46% (P<0.01) and 44% (P<0.05), respectively, using micropositron emission tomography. Plasma FFA concentrations and glycemia, however, were not significantly different between groups.

Conclusion

Our results show that both AG and UAG are cardioprotective against MI/R, but exert different cardiometabolic effects, with UAG protecting against the early increased FA uptake following reperfusion of ischemic hearts. Our study suggests that UAG could be a promising therapy to reduce myocardial ischemia/reperfusion injury.

Supported by the Heart and Stroke Foundation of Quebec and the Canadian Institutes of Health Research.
IWP‐427, an sGC Stimulator, Has Cardioprotective Effects in the Monocrotaline Model of Pulmonary Hypertension
Authors: Jacobson Sarah , Tang Kim , Long Kimberly , Germano Peter , Tobin Jenny , Sheppeck James , Nakai Takashi , Masferrer Jaime
Published: June 2021

Pulmonary arterial hypertension (PAH) results in increased resistance of blood return to the pulmonary vasculature and concomitant hypertrophy of the right ventricle of the heart (RHH). Stimulation of the NO/sGC/cGMP pathway has been shown to reduce RHH in preclinical models of PAH and improves clinical outcomes in patients with PAH. We therefore hypothesized that prophylactic treatment with an sGC stimulator would prevent RHH in a preclinical model of PAH. IWP‐427 is a potent (HEK EC50=167nM), orally bioavailable sGC stimulator. In this study, a single subcutaneous injection of monocrotaline (MCT; 60mg/kg) induced significant RHH in male rats compared to naïve controls after 4 weeks as measured by the ratio of right heart weight to the left ventricle and septum (0.33±0.02 vs 0.22±0.01; p<0.001; n=9‐12). Prophylactic treatment with IWP‐427 prevented MCT‐induced RHH as compared to vehicle treated animals (0.21±0.03 vs 0.33±0.02; p<0.01; n=9‐12). Additionally, plasma levels of NT‐proBNP, a marker of right ventricular damage, were significantly lower in animals treated with IWP‐427 compared to vehicle (10913±1024 vs 15961±1993 ECL units; p<0.05; n=8). IWP‐427 is therefore a cardioprotective sGC stimulator in a monocrotaline model of PAH.
Unholy Fire: Discovering Michael Servetus' Contribution to Pulmonary Physiology Through Modern Historical Fiction
Authors: Didion Sean
Published: June 2021

Unholy Fire is a recent work of modern historical fiction by Don Stratton. Stratton's novel centers in part on the work of Michael Servetus (b. 1511) an anatomist, cartographer, and mathematician whose writings in his theological magnum opus Christianismi Restitutio put him at odds with the Catholic Church as well as John Calvin. Servetus was tried and found guilty for his anti‐Trinitarian views and was burned at the stake in 1553 in Geneva, Switzerland. Exactly who was Michael Servetus and how did his Christianismi Restitutio play an important role in the history of physiology? Despite the mainly theological nature of Servetus' Christianismi Restitutio, he is credited with the first accurate description, including key physiological and mechanical details, of the pulmonary circulation. First, in Christianismi Restitutio Servetus described how the spirit (blood) originates in the left ventricle and with the aid of the lungs is transported through the right ventricle and that this occurs through anastomoses within the lung and not through pores in the cardiac septum as previously postulated by Galen (129‐210 AD). Second, he said that it is in the lungs that the mixture of inspired air occurs and that this mixture contributes to the change in color of the blood. Third, Servetus hypothesized that based simply on the diameter of the arterial vein (pulmonary artery) it is too large in caliber to be for the sole purpose of supplying blood to the lungs. Because of his scholarly acumen, Servetus' placed himself among the first to shed new light on our understanding of the pulmonary circulation thereby loosening the dominance that Galen's teachings had held on medicine for nearly 14 centuries.
The Macrophages as the Stimulators of B‐cells Proliferation in the Pancreas
Authors: Danilova Irina , Bulavintseva Tatiana , Gette Irina , Medvedeva Svetlana , Abidov Mussa
Published: June 2021

The main reason for the development of type 1 diabetes is decrease of the absolute number of B‐cells and their functional activity. The goal of our study was to investigate the regulation of B‐cells regeneration and function.

The model of alloxan diabetes was simulated on male Wistar rats according the author's modified method. The treatment of animals responded EC Directive 2010/63/EU. The content of macrophages was determined by the immunohistochemical study of the pancreatic sections (MAC387) (Abcam), and the number of proliferating B cells was detected with double immunohistochemical staining (insulin antibodies (clone E11D7, Millipore) and Ki67 (BD)). The number and size of pancreatic islets were counted. The derivatives of aminoftalhydrozide were used as the modulators of macrophage activity (patent). The density of B‐cells, their proliferative and secretory activity were significantly decreased of the pancreas of diabetic animals, the average size of islets was also decreased. The number of macrophages in the pancreas islets of diabetic animals was drastically increased. This was reduced by half after immunomodulation. The result of immunomodulation was the increase of islets' size, B‐cells' content and their secretory activity. Consequently, a significant decrease of glucose and Hb1c concentration and an increase of the level of insulin were observed in blood.

These results afford to use the modulation of macrophage as a promising strategy in the stimulation of reparative processes in the pancreas.
Synaptopodin Couples Epithelial Apical Contractions to Adherens Junction Maturation
Authors: Tang Vivian
Published: June 2021

Cell‐cell adhesive contact is a central player in mechanotransduction, providing both mechanical support and a hub for signaling. Yet, the molecular mechanism is poorly defined, partly due to a lack of cell culture model to manipulate tension at cell‐cell contacts. Here, using live cell imaging of α‐actinin, we showed that MDCK cells exhibit many cellular behaviors of epithelial cells in vivo, including (1) oscillating contractility of the junction, (2) pulsatile centripetal cellular contractions, and (3) ratchet constrictions of apical domain. We hypothesized that pulsatile contractions participate in the maturation of junctional complexes. To test our hypothesis, we applied cyclic pulsatile tension to cell‐cell contacts in confluent cell monolayers. Pulsatile intercellular tension induces recruitment of α‐actinin‐4 in a time and tension‐dependent manner without changing the localization of canonical adherens junction proteins, suggesting that a missing factor is involved. We have identified this missing factor as synaptopodin. Synaptopodin recruitment to the junction is also tension‐sensitive, which is necessary for the generation of contractility at cell‐cell adhesions. Recruitment of a‐actinin‐4 strengthens cell‐cell adhesion and promote epithelial permeability barrier. Our studies reveal a molecular system with both mechanical and biochemical inputs and underscore the complexity of junction assembly in epithelial monolayers in vivo and in cultured cell systems.
Substrate Stiffness and Microtopography in PIP2 Regulation of the Actin Cytoskeleton in Primary Cardiac Fibroblasts
Authors: Mkrtschjan Michael , Li Jieli , Russell Brenda
Published: June 2021

Following tissue injury, fibroblasts are one of the primary responding cell types in the wound healing process. Upon myocardial infarction, local fibroblasts divide and increase collagen secretion, leading to cardiac fibrosis. In this study, the effect of the mechanical environment, through substrate stiffness and three‐dimensional topography, is examined in relation to the distribution and levels of phosphatidylinositol 4,5‐bisphosphate (PIP2) in primary neonatal rat ventricular fibroblasts. PIP2 is a membrane component and known regulator of the actin cytoskeleton. Substrates possessing 10 µm high microprojections are fabricated from polydimethylsiloxane to a stiffness of either 100 kPa or 400 kPa (Young's Modulus). A 3‐fold cellular decrease (P<.001) in PIP2 is found between flat‐soft substrates and flat‐polystyrene culture dishes. Additionally, microprojections are shown to blunt PIP2 by a similar amount within each stiffness condition. Fluorescence recovery after photobleaching, yielding the rate constant Kfrap, allows study of actin dynamics in the various micromechanical conditions. A 4‐fold decrease (P<.0001) is found between actin dynamics within lamellipodia to those within stress cables, correlating to a migrating or an anchored cell. This difference in dynamics coincides with an increased redistribution of PIP2 to the lamella in migrating cells. An improved understanding of the intracellular lipid mechanisms involved in wound healing could aid in prevention of maladaptive scarring following major injury. Funded by NIH HL62426, T32HL07692
Shear Stress Induces G Protein‐Coupled Receptor (GPCR)‐Independent Heterotrimeric G Protein Activation in Endothelial Cells
Authors: dela Paz Nathaniel , Melchior Benoit , Frangos John
Published: June 2021

Mechanotransduction is the process by which mechanical forces, such as shear stress, are converted into biochemical responses within the cell. The molecular mechanisms by which endothelial cells (ECs) sense and transduce shear stress into biological signals have not been fully elucidated. G proteins and G protein‐coupled receptors (GPCRs) have been hypothesized independently to mediate mechanotransduction. In this study, we developed a novel read‐out for in situ detection of GPCR/Gαq/11 activation, based on the proximity ligation assay (PLA). We adapted this approach to examine the role of specific GPCR/Gαq/11 pairs in shear stress‐induced EC mechanotransduction. We demonstrated that S1P stimulation causes a rapid dissociation of Gαq/11 from its receptor, S1P3, followed by a dramatic increase in association of S1P3 with both GRK2 and β‐arrestin in human coronary artery ECs (HCAECs), indicative of GPCR/Gαq/11 activation and receptor desensitization/internalization. The S1P3 antagonist, CAY10444, as well as the G protein inhibitor, GDP‐β‐S, prevented this activation in S1P‐treated cells. Interestingly, shear stress led to the rapid dissociation of Gαq/11 from S1P3. However, associations between S1P3 and GRK2 and S1P3 and β‐arrestin were not markedly increased over time. Pre‐treatment of cells with GDP‐β‐S prevented the dissociation of Gαq/11 from S1P3 in response to shear stress. In contrast, treatment of cells with AlF4‐ resulted in increased dissociation of Gαq/11 from S1P3, but no increase in association between S1P3 and either GRK2 or β‐arrestin. Altogether, these findings support the notion that Gαq/11 participates in the sensing/transducing of shear stress independent of GPCR activation in ECs.
INHALATION OF HYDROGEN SULFIDE (H2S) AUGMENTS THE PULMONARY REFLEXES
Authors: Hsu Chun‐Chun , Yu Ching‐Yi , Lin You Shuei
Published: June 2021

We have previously reported that H2S exerted a sensitizing effect on pulmonary vagal C‐fiber afferents in anesthetized, artificially ventilated SD rats. However, whether the sensitizing effect of H2S in turn enhances pulmonary reflexes remains unknown. In anesthetized, spontaneously breathing rats, inhalation of aerosolized sodium hydrosulfide (NaHS, a donor of H2S) caused no significant changes in the baseline breathing pattern. However, the pulmonary chemoreflexes, characterized by apnea, bradycardia and hypotension, triggered by right atrial injection of capsaicin or adenosine, were potentiated after the NaHS inhalation. This potentiating effect was eliminated by pretreatment with perineural capsaicin (a selective blockade of the conduction of vagal C‐fibers) or HC‐030031 (an antagonist of TRPA1 receptors), but not affected by pretreatment with perineural sham surgery or HC‐030031 vehicle. Furthermore, in awake, restrained guinea pigs, the cough reflexes elicited by inhalation of aerosolized capsaicin were also potentiated after the NaHS inhalation. In conclusion, H2S enhanced pulmonary reflexes, and probably resulted from its action on pulmonary C‐fibers. (MOST 103‐2320‐B‐038‐014‐)
Vitamin D Attenuates TNFα‐induced Chemokine Production in Developing Human Airway Smooth Muscle Cells
Authors: Britt Rodney , Faksh Arij , Vogel Elizabeth , Thompson Michael , Pandya Hitesh , Amrani Yassine , Pabelick Christina , Prakash YS
Published: June 2021

Introduction: Children with low Vitamin D levels are at increased risk of developing asthma. In the present study, we tested the hypothesis that the Vitamin D receptor (VDR) agonist, calcitriol, will reduce tumor necrosis factor‐α (TNF‐α)‐induced expression of chemokines in human fetal airway smooth muscle cells (fASM).

Methods and Results

Human fASM cells were dissociated from canalicular stage (18‐22 weeks gestation) lung tissue (de‐identified samples, in vitro use only and considered exempt by Mayo Institutional Review Board). Fetal ASM cells were pre‐treated with vehicle (0.001% DMSO) or 100 nM calcitriol for 1 h, and then exposed to 10 ng/mL TNFα for 6 or 24 h. Expression of CCL5 and CCL11 were measured by quantitative real time PCR and ELISA. Cells were transfected with VDR siRNA. Treatment with TNFα for 6 h increased mRNA expression and secretion of CCL5 and CCL11. While pre‐treatment with calcitriol reduced CCL5 and CCL11 expression in the presence of TNFα. Knockdown of the VDR expression demonstrated that calcitriol's effects are mediated through VDR.

Conclusions

These novel data demonstrate that calcitriol modulates cytokine enhancement of chemokines in developing ASM. Vitamin D may be a new therapeutic target in reducing inflammation in the immature airway.

Supported by NIH F32 HL123075 (Britt), T32 HL105355 (Vogel), and R01s HL088029 and HL056470 (Prakash), and the Mayo Clinic Department of Anesthesiology (Pabelick, Prakash).
The Severity of Lethal H5N1 Viral Infection‐Induced Changes in Pulmonary Inflammation and Functions Is Age‐Dependent
Authors: Zang Na , Zhuang Jianguo , Ye Chunyan , Pollock Zemmie , Tipper Jennifer , Xu Fadi
Published: June 2021

H5N1 viral infection results in ~60% mortality with the highest rate in the juvenile (10‐19 years old). Our previous study has shown that all Balb/c mice at 4W age die 8‐9 days after lethal H5N1 viral (HK483) infection, in which a remarkable drop of body temperature (BT) is an indication of fatality (AJRCCM 2013). This study was undertaken to test the hypothesis that HK483 infection‐induced changes in pulmonary inflammation and function, and drop of BT and body weight (BW) were greater in juvenile (20W) than that in younger (4W) mice. The two age groups of mice were intranasally inoculated with HK483 virus (100 PFU). After measurement of BW and BT, they were anesthetized for collecting arterial blood and then euthanized for BALF, brain and lung tissue collections on days ‐1, 3, 5, and 7 postinfection. We found that the viral infection similarly increased BALF cells, and gradually decreased BW, BT, lung dry/wet ratios, and Sao2 in the two groups without difference in lung H5N1 viral titers. At the early stage of infection the dominant increases of BALF cell types were neutrophils in 4W but lymphocytes in 20W mice. At late stage, 20W mice, compared to 4W mice, showed less severe BT/BW drops and a two‐day delay in the occurrence of hypoxemia and hypercapnemia (appeared on day 5 postinfection). Our data suggest that the impact of lethal H5N1 viral infection on pulmonary inflammation and function is age‐dependent, i.e., it is worse in younger than juvenile mice, which is somewhat different from the clinical observations in humans (Supported by HL119683).
Hypercontractile Airway Smooth Muscle is a Potential Driver of Respiratory Pathophysiology in Congenital Muscular Dystrophy 1A
Authors: Pate Jessica , Kumar Ajay , Girgenrath Mahasweta , Morgan Kathleen
Published: June 2021

Merosin deficient congenital muscular dystrophy (MDC1A) is a fatal, autosomal recessive disease caused by laminin α2 chain deficiency, preventing formation of the extracellular matrix protein laminin 211. Afflicted children experience profound muscle weakness and degeneration and often die prematurely from failure to thrive or acute respiratory failure resulting from weak respiratory muscles and progressive scoliosis. While diseased skeletal muscle has been well characterized, airway smooth muscle (ASM) has not been investigated and respiratory failure has been attributed solely to diaphragmatic weakness. Therefore, as a first step we investigated the contractile properties of ASM isolated from DyW mice, an animal model of MDC1A.

Primary bronchial rings from 7wk WT and DyW mice were harvested and cleaned. Rings were mounted on wire triangles in a bath with oxygenated Krebs solution and connected to a force transducer to measure contractile responses to depolarizing 51mM KCl Krebs solution and 10‐5 M carbachol to activate G‐protein coupled muscarinic receptors. Rings were quick frozen and saved for histological analysis. Transverse sections (7 µm thick) were stained with Gomori Trichrome to differentiate ASM. Bronchial ASM cross sectional area (CSA) was calculated using NIS Basic Research software. Contractile forces were normalized to ASM CSA to determine ASM stress.

The stress responses of DyW bronchi are significantly higher than WT in response to both 51 mM KCl (nWT=7, nDyW=3, p<0.002) and 10‐5 M carbachol (nWT =8, nDyW =4, p<0.006). These data indicate that ASM isolated from DyW mice are significantly hypercontractile compared to their WT counterpart, and could potentially contribute to respiratory insufficiency in MDC1A.
Role of PGE2/EP4 Receptor Signaling on The Effects of Hyperoxia on Airway Smooth Muscle
Authors: Jakupaj Muharrem , Sopi Ramadan , Dreshaj Ismail , Mladenov Mitko , Temaj Gazmend , Cenaj Asllan , Neziri Burim , Beqiri Qëndresa , Krasniqi Avdyl
Published: June 2021

It was shown that neonatal hyperoxic‐exposure associates with airway hyperresponsiveness and impaired nonadrenergic noncholinergic relaxation. It was demonstrated that reduced relaxant responses were associated with an impairment of prostaglandin E2 (PGE2) release from airway smooth muscle (ASM). In this study we tested the hypothesis that supplementation of PGE2 or EP4 agonists to ASM restore relaxant responses. Tracheal cylinders were obtained from 12 days rat pups exposed to hyperoxia or room air for seven days. These cylinders were used to study contractile and relaxant responses. In hyperoxic trachea the contractile responses to bethanechol were enhanced as compared to room air controls. These enhanced contractile responses were attenuated by pre‐incubation of tissues with PGE2 (1µM). Similar effect showed an EP4 agonist. This effect of PGE2 and EP4 agonist was reversed by an EP4 antagonist. PGE2 and EP4 induced dose response relaxations of ASM, which were reduced by an EP4 antagonist.

We conclude that PGE2 induces relaxation of airways via EP4 receptors, and the use of EP4 agonists may serve as a therapeutic approach to overcome the adverse effects of neonatal hyperoxia on relaxant responses of ASM.

Supported by MEST
Influence of Maturation on Ca2+ Waveform Modulation by c‐AMP and c‐GMP in Pulmonary Arterial Smooth Muscle of Sheep
Authors: Paez Ricardo , Romero Monica , Dobyns Abby , Francis Michael , Taylor Mark , Longo Lawrence , Wilson Christopher , Wilson Sean
Published: June 2021

One critical feature of the pulmonary vasculature is the ability to vasodilate in response to PKA and PKG signaling, which are important at birth and later in life. These kinases are activated by cAMP and cGMP respectfully, which can modulate Ca2+ release through enhanced activity of ryanodine receptors (RyRs) and Sarco/Endoplasmic Reticulum Ca2+ ‐ATPase (SERCA) pumps. To test the relevance of maturity on unstimulated Ca2+ waveforms and determine the effects of c‐AMP and c‐GMP on their function, we examined the influence of 3‐isobutyl‐1‐methyl xanthine (IBMX), a PDE inhibitor that primarily increases c‐AMP, as well as 8‐Br‐c‐AMP and 8‐Br‐c‐GMP that are degradation resistant c‐AMP and c‐GMP isoforms. Cytosolic Ca2+ wave activity was measured in fetal and adult sheep via confocal Ca2+ imaging of individual myocytes in artery strips. Maturation increased the percentage of responsive cells independent of altitude while IBMX, 8‐Br‐c‐AMP, and 8‐Br‐c‐GMP each failed to alter the number of active myocytes in both groups. c‐GMP increased net Ca2+ wave activity through temporal changes while IBMX and c‐AMP had minimal effect on Ca2+ activity. Yet, c‐AMP increased the interaction between regions of interest (ROIs) in adults but not fetuses. The firing of ROIs near to one another was enhanced by cAMP in adults but reduced in fetuses, while c‐GMP increased this in both. Even though future research is required to fully comprehend the role of Ca2+ signals during normal cellular maturation, our results suggest that c‐GMP amplifies these Ca2+ signals.
Hypoxia Decreases Trx2 Protein and Activity in Pulmonary Vascular Cells
Authors: Wade Brandy , Adesina Sherry , Hart Michael , Sutliff Roy
Published: June 2021

Pulmonary Hypertension (PH) is characterized by the sustained increase in pulmonary arterial pressure. Increased pulmonary vascular pressure and resistance result in right ventricle (RV) hypertrophy and can ultimately lead to RV failure and death. Increased levels of Reactive Oxygen Species (ROS) have been implicated in PH pathogenesis. Endogenous antioxidant mechanisms, such as the Thioredoxin 2 (Trx2) system, are of importance in maintaining cellular ROS levels. Trx2 protein acts as a reducing agent within the mitochondria to maintain proteins in a reduced state. Because hypoxia stimulates ROS and PH, we examined the effects of hypoxia on Trx2 in vitro and in vivo. We hypothesized that hypoxia impacts mitochondria ROS levels by altering Trx2 expression and activity. Human Pulmonary Artery Endothelial Cells (HPAEC) were exposed to hypoxia (1%O2) for 72 hours, and mice were subjected to 3 weeks of hypoxia (10% O2). qRT‐PCR and western blot analysis were used to study the transcriptional, translational and post‐translational effects of hypoxia on Trx2. Hypoxia decreased levels of Trx2 mRNA and protein in HPAEC in vitro and also reduced Trx2 mRNA levels in the lungs of hypoxia‐exposed mice. Similarly, hypoxia increased oxidized Trx2 (inactive) in the mouse lung compared to animals exposed to normoxia. In combination these data suggest that hypoxia decreases in vitro and in vivo. These changes in Trx2 expression and activity may contribute to mitochondrial ROS production and dysfunction associated with PH. This work was supported by NIH RO1 HL 102167 and the T32 Training Program in Academic Pulmonary Medicine.
HIF Upregulates AQP1 Expression in Pulmonary Arterial Smooth Muscle Cells (PASMCs) in a Calcium‐Dependent Manner
Authors: Yun Xin , Maman Stephan , Jiang Haiyang , Shimoda Larissa
Published: June 2021

Prolonged exposure to hypoxia can have adverse effects on the pulmonary vasculature, including structural remodeling and sustained contraction, resulting in the development of pulmonary hypertension. Both PASMC proliferation and migration contribute to the vascular remodeling. The expression of AQP1, a membrane water channel protein, is elevated during hypoxia. Studies in other cell types suggest that AQP1 is a direct transcriptional target of hypoxia inducible factor (HIF)‐1. Moreover, an increase in intracellular calcium ([Ca2+]i) concentration is a hallmark of hypoxic exposure in PASMCs. Thus, we wanted to determine whether HIF regulates AQP1 in PASMCs and, if so, whether the process was Ca2+‐dependent. PASMCs were incubated with DMOG, which inhibits HIF protein degradation. DMOG produced a time‐dependent increase in AQP1 protein, but not mRNA. Interestingly, incubation with DMOG increased [Ca2+]i in PASMCs, and this elevation was prevented by the voltage‐gated Ca2+ channel inhibitor, verapamil (VER). VER also blocked upregulation of AQP1 protein by DMOG, but had no effect on expression of GLUT1, a canonical HIF transcriptional target. Thus, our results show that contrary to reports in other cell types, in PASMCs, AQP1 does not appear to be a direct target for HIF transcriptional regulation. Instead, AQP1 protein may be upregulated by a mechanism involving HIF‐dependent increases in [Ca2+]i.
Serotonergic modulation of Locus coeruleus (LC) neurons in the newborn rat
Authors: Biancardi V , Alvares T , Gargaglioni L , Funk G
Published: June 2021

Serotonergic (5HT) and noradrenergic (NA) neurons project throughout the CNS where they influence brain development and modulate many processes including breathing. The degree to which these modulatory systems interact is unknown. However, 5HT is released in the LC, a NA nucleus involved in central chemosensitivity. Also, exposure of rats to 5HT reuptake inhibitors (SSRIs) perinatally results in adults with hyperexcitable LC neurons. As a significant number of pregnant women take SSRIs, this study aimed to: (1) investigate the effects of perinatal fluoxetine on the ventilatory responses of newborn rats to 7% CO2; (2) assess in naïve newborn rats the mechanisms by which 5HT affects the excitability of LC neurons. Neonates exposed to fluoxetine (E15‐21) had a hypercapnic tidal volume response 31% greater than control. Of 150 whole‐cell voltage‐clamped LC neurons, 101 responded to 5HT (100µM) with an inward current (‐30±3 pA), 18 responded with oscillations (freq of 0.4±0.1 Hz), and 31 did not respond. 5HT3R agonists had no effect. 5HT2R agonists (PNU22394 & TCB‐2, 100µM) evoked 3 responses: an 822% increase in the freq of glutamatergic mEPSCs (29/37 neurons); an inward current (‐15±2 pA; 22% increase in RN, 16/37); and a nitrendipine‐sensitive 0.1 Hz oscillation (5/37). The 5‐HT1AR agonist 8‐OH‐DPAT (50µM) induced an inward current (‐14±3 pA, 6/11), while Sumatriptan, a 5HT1ABDR agonist had no effect. These data demonstrate that perinatal SSRI exposure alters respiratory chemosensitivity. Part of this effect may reflect diverse actions of 5HT on LC neurons that include a presynaptic 5HT2‐mediated increase in glutamatergic EPSCs, and postsynaptic facilitation of Ca2+‐dependent membrane oscillations and a 5HT2/5HT1A‐ inward current. Support: CIHR, WCHRI, AIHS, CNPq, FAPESP.
Developmental Nicotine Exposure Results in Exaggerated Response to AMPA Receptor Activation in Hypoglossal Motoneurons in Neonatal Rats
Authors: Cholanian Marina , Levine Richard , Fregosi Ralph
Published: June 2021

Nicotine is the primary neurotoxin in tobacco products, as it has been strongly associated with altered development of neurons throughout the brain, including brainstem neurons involved in the control of breathing. Chronic nicotine exposure causes widespread desensitization of nicotinic acetylcholine receptors (nAChRs), leading to increased receptor expression, but a reduction in synaptic efficacy. Our previous work in rhythmically active hypoglossal motoneurons (XIIMNs) showed that developmental nicotine exposure (DNE) desensitizes their nAChRs and reduces excitatory synaptic input to these cells. We also showed that expression of somatic glutamate receptors was reduced, suggesting a reduction in post‐synaptic efficacy. Here we hypothesize that DNE results in increased postsynaptic responses to AMPA receptor activation in XIIMNs from neonatal rats. Whole cell voltage clamp recordings of rhythmically active XIIMNs in brainstem slices from control and DNE animals were made. AMPA (2.5 mM) was bath‐applied in the presence of TTX to isolate fast postsynaptic glutamatergic responses. Preliminary data suggest a larger AMPA‐mediated inward current and a higher charge transfer in DNE XIIMNs (DNE, n = 10; control, n = 12). All responses were blocked by CNQX (20 mM), a selective AMPA and kainate receptor antagonist, indicating that the responses were AMPAergic. These results indicate that DNE: (1) upregulates AMPA receptor expression in XIIMNs, and/or (2) changes the kinetics of AMPA receptors in order to overcome the reduced excitatory synaptic input to XIIMNs.
Desensitization of mu opioid receptors on Kölliker‐Fuse neurons
Authors: Levitt Erica , Abdala Ana , Paton Julian , Williams John
Published: June 2021

Tolerance to the respiratory depressant effects of opioids develops less readily than tolerance to analgesia, creating a dosing limit in chronic pain patients. Desensitization of mu opioid receptors is considered an initial step in the development of tolerance. Currently, most information about desensitization has been obtained in locus coeruleus (LC) neurons. Given the lack of respiratory tolerance, this study examined desensitization of mu opioid receptors on respiratory‐controlling Kölliker‐Fuse (KF) neurons. In voltage‐clamp recordings from KF neurons in rat brain slice, the opioid receptor agonist [Met5]enkephalin (ME) caused an outward current in a population of KF neurons due to activation of GIRK conductance. Compared to ME, the partial opioid agonist morphine produced a current that was proportionally larger than the morphine‐mediated current in LC brain slice preparations, indicating that KF neurons may have a large receptor reserve and thus be resistant to desensitization. Desensitization was induced by prolonged (10 min) perfusion of a high concentration of ME (30 µM). Desensitization of the ME‐mediated GIRK current was significantly less in KF neurons than in LC neurons. In contrast to LC, desensitization was not enhanced by activation of PKC or in slices from morphine‐treated rats. Thus, a lack of desensitization of mu opioid receptors on KF neurons may contribute to the relative lack of tolerance to the respiratory depressant effect of opioids. This will be tested using in situ and in vivo preparations of morphine‐treated rats. Supported by DA038069 (ESL) and DA08163 (JTW).
Atypical respiratory Physiology of a recent air‐breathing fish: Pangasius
Authors: Bayley Mark , Phuong My , Damsgaard Christian , Huong Do Thi Thanh , Wang Tobias
Published: June 2021

Air‐breathing in fish has evolved independently many times and is normally associated with marked reduction in gill size, often argued as an adaption to avoid oxygen loss to hypoxic water, while retaining a capacity for branchial ion and acid‐base regulation. This is not the case in the economically important, but sparsely studied teleost Pangasius (Pangasionodon hypophthalmus), which seems to have acquired air‐breathing during a recent a speciation event in the mid‐Miocene. Using a heavily vascularized and modified swim bladder, it has retained well‐developed gills that can provide for an almost five‐fold rise in oxygen uptake from normoxic water during swimming without access to air. Its gills, however, show considerable plasticity, such that the secondary lamella become almost completely embedded by an interlamellar when the fish are kept in normoxic water, becoming fully exposed in hypoxic water. The blood and isolated hemoglobin of Pangasius have extremely high oxygen affinities with P50′s of a mere 4‐6 mm Hg at 30oC, but there is no evidence for a Root effect and the red cells are devoid of adrenergic stimulation of the sodium‐proton exchanger. Moreover, as an additional difference to other air‐breathing fishes, Pangasius shows efficient extracellular pH regulation and can be found growing in strongly hypercapnic water with PCO2 in excess of 40 mm Hg. Based on these unusual attributes in an air‐breathing fish, we will present data on the regulation of cardiac and ventilator responses to hypoxia from fish acclimated to different temperatures and oxygen regimes.
Impact of Mineralocorticoid, Glucocorticoid and Thyroid Hormones on Respiratory Control Development in Lithobates Catesbeianus
Authors: Rousseau Jean‐Philippe , Dédé Djigo Aicha , Kinkead Richard
Published: June 2021

The emergence of air breathing during Lithobates catesbeianus development requires significant changes to the brainstem circuits that generate and regulate breathing; however, the mechanisms responsible for this transformation remain largely unknown. Because this metamorphosis is regulated by hormones such as corticosterone, aldosterone, and thyroid hormone (T3), we tested the hypothesis that exposing the brainstem to these hormones augments the fictive air breathing frequency in Lithobates tadpoles. Brainstems were isolated from pre‐metamorphic tadpoles (TK V to X) and placed in a bottle containing artificial cerebrospinal fluid and one of the following hormones: aldosterone (100nM), corticosterone (100nM), T3 (100 nM) or a combination of two of the preceding components for 24 hours. The effect of hormone exposure on respiratory activity was quantified by extracellular recording of rhythmic motor output from cranial nerves V and X. By comparison with preparations subjected to sham treatment, hormone exposure generally increased fictive air breathing frequency; the highest lung burst frequency was observed in brainstems exposed to the aldosterone‐T3 combination. To assess the treatment effects on the overall system functionality, sham and hormone treated tadpoles were kept in a recording chamber for 24h after which we recorded their ventilator activity by measuring changes in buccal pressure. Interestingly, the results showed the same trend as the one observed with the brainstem preparation. We conclude that hormones play an important role in the maturation of the neural circuits that generate and regulate breathing in this species.