bluuebunny/crossref_metadata_2025 huggingface dataset:
A Mathematical Model of Calcium and cAMP Signaling in Pulmonary Microvascular Endothelial Cells
Authors: Webb Kristal J , Wiles C Alex , Annamdevula Naga , Sweat Rachel , Britain Andrea L , Phan Anh‐Vu , Townsley Mary I , Leavesley Silas J , Rich Thomas C
Published: November 2023

The goal of this project is to understand how the spatial distribution of intracellular Ca2+ alters the kinetics and spatial spread of agonist‐induced cAMP signals in pulmonary microvascular endothelial cells (PMVECs). PMVECs predominantly contain the Ca2+‐inhibited adenylyl cyclase type 6 (AC6) and also contain low levels of the Ca2+‐stimulated phosphodiesterase type 1 (PDE1). It has been clearly demonstrated that Ca2+ influx through store operated Ca2+ (SOC) channels inhibits AC6 activity in PMVECs. Our preliminary data indicate that Ca2+ influx through T‐type Ca2+ channels and TRPV4 cation channels inhibits agonist‐induced cAMP production. We have developed mathematical models to investigate how the spatial reach of Ca2+ influx through each of these channels alters agonist‐induced cAMP signals. The models describing this intracellular feedback network were developed in the MATLAB programming environment. Our initial models used ordinary differential equations to describe the kinetics and spatial redistribution of Ca2+ and cAMP signals between intracellular compartments. Based upon experimental observations, Ca2+ channels were distributed in three discrete subplasmalemmal compartments. In addition, a fourth cytosolic compartment was incorporated into the model. This model also describes Ca2+ and cAMP buffering as well as Ca2+ efflux. A Runge‐Kutta solver was used to perform numerical integration. Simulations of the model predict that at low agonist levels, low levels of Ca2+ influx, Ca2+ signals do not readily spread between compartments. Similarly, the model predicts that agonist‐induced cAMP signals do not readily spread between compartments. Thus, Ca2+ influx in one compartment leads to localized inhibition of AC6 and a localized decrease in cAMP. We conclude that at low agonist concentrations, activation of different populations of Ca2+ channels regulates different pools of AC6 thus cAMP levels in different subcellular compartments. However, at higher and/or prolonged agonist concentrations, Ca2+ signals spread between intracellular compartments, increasing the range of AC6 inhibition. We are currently extending this model to more accurately describe the relative diffusion of second messengers using a finite element analysis approach.

Support or Funding Information

This work was supported by NIH P01HL066299 and T32HL076125.
A‐Kinase Anchoring Proteins Coordinate the Contractile Phenotype of Airway Smooth Muscle
Authors: Poppinga Wilfred Jelco , Han Bing , Elzinga Carolina R. , Halayko Andrew J. , Meurs Herman , Schmidt Martina
Published: November 2023

Smooth muscle contraction is modulated by three pathways: the amount of free [Ca2+]i; Ca2+ sensitivity; and, the regulation of intracellular filaments comprising the contractile machinery. Recently, a new dimension has emerged to play a role in airway smooth muscle function, namely the scaffolding protein family A‐kinase anchoring proteins (AKAPs) that bind cAMP effector PKA and other proteins to specific cellular compartments. Here we studied the role of AKAPs in maintaining the contractile phenotype of the airway smooth muscle. Treatment with AKAP‐PKA interaction inhibitor st‐Ht31 (50 μM) for 96 h caused an increase in abundance of the contractile proteins α‐smooth muscle actin (αSMA) (283% of basal) and calponin (159% of basal) in immortalized human tracheal smooth muscle cells, without an increase in mRNA expression or stability. To understand potential underlying mechanisms for this response, after 72 h incubation with st‐Ht31, we treated cells (24 h) with the mRNA transcription inhibitor, actinomycin D (1 μg/mL): this decreased abundance of both proteins in absence and presence of st‐Ht31 (~50 αSMA, ~70% calponin). Nonetheless, st‐Ht31 still induced higher protein levels compared to untreated control cultures (~35% αSMA, ~80% calponin). Inhibition of protein synthesis using cycloheximide (5 μg/mL) had no effect on the expression αSMA, however it did increase the expression of calponin under both control and st‐Ht31‐treatment conditions (~200%), suggesting that αSMA is a rather stable protein and that calponin degradation requires de novo synthesis of an unknown protein. Lysosomal inhibition using chloroquine (50 μM) did not affect either protein. Inhibition of the proteasome with MG‐132 (5 μM) reversed st‐Ht31‐induced increase of αSMA (102% of basal), without affecting the st‐Ht31 induced increase of calponin (210% of basal), revealing that st‐Ht31‐induced increase in αSMA requires proteasomal degradation of an unknown protein. Functionally, incubation of human tracheal smooth muscle strips with st‐Ht31 for 96 h increased both the abundance of contractile proteins and the contractile response to methacholine. These data implicate that AKAPs regulate the maintenance of contractile smooth muscle, probably by post‐translationally controlling levels of contractile proteins.

Support or Funding Information

This study was supported by the Dutch Lung Foundation (Grant #: 3.2.11.015) and an Ubbo Emmius grant.
Bayesian Analysis of E3 Ubiquitin Ligase/AQP2 Interactions in the Renal Collecting Duct
Authors: Medvar Barbara L , Sarkar Abhijit , Knepper Mark
Published: November 2023

Aquaporin‐2 (AQP2) is regulated in part via vasopressin‐mediated changes in protein half‐life that are in turn dependent on AQP2 ubiquitination. Here we addressed the question, “What E3 ubiquitin ligase is responsible for AQP2 ubiquitination?” using large‐scale data integration based on Bayes’ Rule. The first step was to bioinformatically identify all E3 ligase genes coded by the human genome. This analysis used the software ABE (Automated Bioinformatics Extractor) to identify all proteins with HECT, RING‐Finger or U‐box domains and combined the resulting list with data from existing databases. The 377 E3 ubiquitin ligases identified in the human genome have been used to create an online database. Bayes’ analysis ranked these 377 E3 ligases regarding probability of interaction with AQP2 using (1) expression data (the mouse mpkCCD transcriptome and the mouse mpkCCD proteome), (2) co‐localization data of E3 ubiquitin ligases with AQP2 in mpkCCD subcellular fractions (quantitative proteomic data), (3) presence of E3 ligases in AQP2‐expressing tubule segments (rat single‐tubule RNA‐seq data), (4) annotation from UniProt records regarding cytoplasmic versus nuclear localization and (5) likelihood of membrane association based on presence of membrane‐spanning domains or phospholipid interaction domains. Application of Bayes’ Rule to these data identified the top ranked E3 ligases from the analysis (in order of probability): NEDD4=NEDD4L>AMFR>STUB1>ITCH>ZFPL1. Three of these proteins (Nedd4, Nedd4‐2 and Itchy) are HECT domain proteins. Prior phosphoproteomic analysis has revealed that Nedd4‐2 undergoes a marked increase in phosphorylation in response to vasopressin in the region that mediates protein‐protein interactions (multi‐WW domain region), suggesting a mechanism whereby AQP2 ubiquitination could be regulated.
N‐cadherin adhesion modulates RhoGTPase signaling to promote endothelial barrier integrity
Authors: Kruse Kevin Jonathan , Huang Fei , Ying Sun , Vogel Stephen , Malik Asrar , Komarova Yulia
Published: November 2023

The vascular endothelium is comprised of a monolayer of endothelial cells (ECs) lining the blood vessels, which functions as a semi‐permeable barrier between blood and the surrounding tissue. Vascular Endothelial (VE)‐cadherin, which forms homotypic adhesion between ECs at cell‐cell contacts called adherens junctions (AJs) is the main adhesive molecule that restricts passage of protein rich fluids through the endothelial monolayer. Along with VE‐cadherin, ECs express Neural (N)‐cadherin, which is excluded from EC‐EC junctions and instead forms heterotypic adhesions between ECs and mural cells (such as pericytes or smooth muscle cells) at the abluminal surface of the endothelium. While the role of VE‐cadherin adhesion in regulating endothelial barrier function has been well established, the role of N‐cadherin in ECs remains mainly unknown. Recent evidence suggests that pericytes are critical to maintaining proper endothelial barrier function, however it is still unclear if this is solely due to pericyte recruitment to the endothelium or if N‐cadherin adhesion mediated signaling in ECs plays a role in strengthening the endothelial barrier. Using an EC specific, inducible knockout mouse model developed in our lab, we have shown that deletion of Cdh2 gene (N‐cadherin) results in increased permeability of pulmonary endothelial microvessels, suggesting that N‐cadherin adhesion restricts permeability in the endothelial barrier. To study N‐cadherin adhesion‐mediated signaling in endothelial monolayers, we have utilized surface chemistry techniques to mimic N‐cadherin homophilic interaction in vitro. Covalent attachment of N‐cadherin extracellular domain to an Ni‐NTA surface in an oriented manner induced formation of N‐cadherin adhesions proximal to tricellular junctions and promoted recruitment of VE‐cadherin to AJs. Depletion of N‐cadherin in ECs or denaturation of N‐cadherin extracellular domain abolished this effect, suggesting that N‐cadherin “outside‐in” signaling promotes assembly of VE‐cadherin adhesion. Using microscale biomimetic platforms combined with mass spectroscopy, we isolated and analyzed N‐cadherin complexes. We found that a dual Rac1 and RhoA activator, Trio, is recruited to N‐cadherin adhesion complexes to activate both Rac1 and RhoA signaling at AJs. This was consistent with increased acto‐myosin tension across VE‐cadherin analyzed with a FRET‐based biosensor as well as formation of lamellipodia protrusions above AJs. Furthermore, inhibition of ROCK signaling or depletion of Neuronal navigator 1 (Nav1) abolished recruitment of VE‐cadherin to AJs specifically in cells plated onto N‐cadherin but not gelatin‐coated surfaces. These data cumulatively demonstrate that N‐cadherin promotes assembly of VE‐cadherin adhesion by providing spatial control of RhoA and Rac1 activities through activation of Trio.

Support or Funding Information

NIH R01 HL103922
Na‐driven Cl/HCO3 exchanger (NDCBE) activity is essential for establishing new steady‐state in intracellular pH during metabolic acidosis in murine hippocampal neurons and astrocytes
Authors: Salameh Ahlam , Boron Walter F
Published: November 2023

The Na‐driven Cl/HCO3 exchanger (NDCBE) is highly expressed in CA1 hippocampal (HC) neurons but has not been reported to be present in astrocytes. NDCBE extrudes acid from the cytoplasm by mediating the exchange of 1 Na+ and 2 HCO3− (or 1 CO3=) from the extracellular compartment for 1 Cl− from the intracellular compartment. Previously, we showed that knocking‐out anion exchanger 3 (AE3), which is an acid loader expressed only in neurons but not astrocytes, reduces the ability of HC neurons and astrocytes to acid load the cytoplasm. We also showed that, after 70 sec in metabolic acidosis (MAc), intracellular pH (pHi) in neurons and astrocytes from AE3−/− mice decreases continuously over 7 min (i.e., fails to stabilize). We hypothesized that this failure to reach new steady‐state is due to a decreased ability of neurons and astrocytes to increase their acid‐extrusion rate (JE) sufficiently to balance the acid‐loading rate (JL). In the present study we investigate the role of NDCBE activity in facilitating sufficient JE to balance JL during MAc. We induced MAc by decreasing the extracellular [HCO3−] from 22 mM to 14 mM at fixed 5% CO2. This maneuver decreases extracellular pH from 7.4 to 7.2. We monitored the changes in pHi during MAc exposure using fluorescence imaging of the pH sensitive dye BCECF. We obtained co‐culture of neurons and astrocytes from the hippocampal CA1 regions from wild‐type (WT) and NDCBE‐knockout (NDCBE−/−) mice. Our results show that, for neurons, the initial steady‐state pHi is significantly higher in WT (7.25 ± 0.03) compared to NDCBE−/− (7.01 ± 0.01). However, the MAc‐induced acidification rate during the first 70 sec of the MAc (dpHi/dt)early is not significantly different between WT (−1.6 × 10−3 ± 0.8× 10−3 pH/s) and NDCBE−/− (−1.4 × 10−3 ± 0.9 × 10−3 pH/s). After 70 sec in MAc, the pHi of NDCBE−/− decreases continuously over 7 min, with a slope, (dpHi/dt)late, of −3.0 × 10−4 ± 0.6 × 10−4 pH/s, which is significantly faster than WT neurons (−0.9 × 10−4 ± 0.6 × 10−4 pH/s). For astrocytes, the knockout of NDCBE surprisingly reduces initial steady‐state from 6.99 ± 0.01 for the WT to 6.90 ± 0.01 for NDCBE−/− and significantly reduces (dpHi/dt)early from −3.3 × 10−3 ± 0.2 × 10−3 pH/s in the WT to −2.2 × 10−3 ± 0.1 × 10−3 in NDCBE−/−. The results also reveal that the absence of NDCBE reduces the astrocytes ability to stabilize pHi after 70 sec in MAc, with a (dpHi/dt)late of −2.0 × 10−4 ± 0.3 × 10−4 pH/s compared to the WT (dpHi/dt)late of −0.6× 10−4 ± 0.6 × 10−4 pH/s. Our results are consistent with the hypothesis that NDCBE activity is essential for braking the fall of pHi in both HC neurons and astrocytes during MAc exposure.

Support or Funding Information

R01‐DK081567, R01‐NS018400 and T32 HL007913
Dysregulation of Pendrin and NBC‐e1 and Impaired HCO3− Secretion in the Kidneys of CFTR Knockout Mice: Role in the pathogenesis of metabolic alkalosis in cystic fibrosis
Authors: Kia Mujan Varasteh , Barone Sharon Lynn , Xu Jie , Zahedi Kamyar , Soleimani Manoocher
Published: November 2023

Introduction

Patients with cystic fibrosis (CF) are prone to the development of metabolic alkalosis, which is manifested by an elevation of arterial blood pH and bicarbonate concentration and produces a physiologic diminution of respiratory effort that is detrimental in CF patients. The pathogenesis of metabolic alkalosis in CF remains unknown.

Hypothesis

Inactivation of CFTR will impair the ability of the kidney to excrete excess bicarbonate via dysregulation of HCO3− transporters, therefore resulting in the elevation of arterial pH and bicarbonate concentration and subsequently, generation of metabolic alkalosis in mutant mice subjected to an oral bicarbonate load.

Methods

CFTR knockout (CF) mice with the intestinal rescue (over‐expressing wild type CFTR transgene in their intestine‐Science 1994) were examined for the changes in arterial pH and bicarbonate concentration as well as the expression of pendrin and NBC‐e1. Animals were subjected to an oral bicarbonate load (140 mM/lit bicarbonate in the drinking water) and examined after 3 days.

Results

Baseline parameters, including acid‐base status and urine pH were comparable in CF and WT mice. Compared to WT animals, CF mice demonstrated a significant increase in blood HCO3− concentration (21.87 +/− 0.8 in wt vs. 26.83 +/− 0.9 mEq/l in CF mice, p<0.004) and pH (7.33 +/− 0.01 in wt vs. 7.42 +/− 0.02 in CF mice, p<0.003) and impaired kidney HCO3− excretion (urine pH 8.10 +/− 0.09 in wt vs. 7.35 +/− 0.09 in CF‐KO mice, p<0.002) following a 3‐day oral bicarbonate load. Immunofluorescence labeling demonstrated profound reduction in the apical expression and increased cytoplasmic and basolateral localization of pendrin in the intercalated cells in CF mice (Fig. 1). Moreover, an unexpected aberrant induction of the Na+:HCO3− co‐transporter NBCe1 (kidney NBC1) was detected in principal cells of the cortical collecting duct in CF mice (Fig. 2).

Conclusion

We propose that patients with cystic fibrosis are prone to the development of metabolic alkalosis secondary to the dysregulation of pendrin and NBC‐e1, specifically during volume depletion, which is a common occurrence in CF patients.

Support or Funding Information

Merit Review Award from Veterans Administration
Structure ‐ function relationship of the human mitochondrial thiamin pyrophosphate transporter (MTPPT; SLC25A19): Critical roles for Ser34, Lys 291 and His137
Authors: Sabui Subrata , Subramanian Veedamali S , Ghosal Abhisek , Kapadia Rubina , Said Hamid M
Published: November 2023

The water‐soluble vitamin B1 (thiamin) plays critical role in cellular energy metabolism. Mammalian cells lack the ability to synthesize thiamin, and thus, must obtain the vitamin from their surrounding via transport across cell membrane. Once in the cytoplasm, free thiamin is enzymatically converted to thiamin pyrophosphate (TPP); the bulk of this TPP is then taken up by the mitochondria by a carrier‐mediated process that involves the MTPPT (product of SLC25A19 gene). Previous studies from our laboratory and others have characterized different physiological and biological aspects of the MTPPT, but little is currently known about its structure‐function relationship. Using protein‐docking modeling (www.sbg.bio.ic.ac.uk/phyre2 and http://www.dockingserver.com/web) and site‐directed mutagenesis, we examined in the current investigation the role of amino acid residues that are predicted (by the docking model) to be involved in the function of the human MTPPT (hMTPPT; i. e., Thr29, Arg30, Ile33, Ser34, Asp37 and Phe298); we also examined the role of conserved (among species) positively charged amino acids (i. e., His82, His137, Lys231 and Lys291) in hMTPPT function of uptake of the negatively charged TPP. Our results showed that of the six predicted residues, Ile33, Ser34, Asp37 appeared to be critical for hMTPPT function as their individual mutation led to a significant reduction in carrier‐mediated MTPP uptake (with no change in protein stability/translational efficiency of the transporter). The cause of impairment in the case of Ile33 and Asp37 appeared to be due to inefficient delivery of the mutant protein to the mitochondria as indicated by confocal imaging and by Western blot analysis. With regards to the role of the conserved positively charged residues, only His137 and Lys291 were found to be important for hMTPPT function as indicated by the inhibition in MTPP uptake upon their mutation. This inhibition in MTPP uptake was again found to be due to inefficient delivery of the protein to the mitochondria (again with no change in protein stability/translational efficiency of the transporter). Results of this investigation show an important role for Ser34, H137 and Lys291 in the function of the hMTPPT.

Support or Funding Information

Supported by grants from the NIH (AA018071 and DK56061) and the DVA
Effects of Varied Ionic Concentrations on The Feeding Ability of Cordylophora caspia, An Invasive Hydroid
Authors: Cruise Jessica D. , Hofer Sarah J. , Busch Jennifer L. , Folino‐Rorem Nadine C.
Published: November 2023

The objective of our study was to observe the effects of KCl concentrations on nematocyst functionality as it related to feeding ability in the invasive hydroid Cordylophora caspia (phylum Cnidaria, class Hydrozoa). Nematocysts are stinging organelles encapsulated in cnidocytes on the tentacles. When tentacles are tactilely stimulated by prey, cnidocytes experience a rapid uptake of water and ions, resulting in a buildup of osmotic pressure that ultimately forces the nematocyst from the cell, rendering the prey immobile. This discharge mechanism makes nematocyst function potentially susceptible to variation in environmental NaCl and KCl concentrations.

Cordylophora caspia can thrive in a variety of osmotic environments, from freshwater to brackish habitats. We assessed the effects of different ionic environments on nematocyst discharge by adding 5 mM KCl to fresh and brackish water. Two genotypes of Cordylophora caspia (one native to fresh water and one native to brackish water) were collected and cultured in the laboratory. The freshwater genotype was native to a salinity of 10 mM NaCl (0.6 ppt), and the brackish genotype was native to a salinity of 270 mM NaCl (16 ppt). Colonies from each genotype were cultured at their native salinity (controls) and at the native salinity of the other genotype in the absence and presence of additional 5 mM KCl.

Our results showed that treatment‐induced effects varied as a function of salinity, rather than genotype. When compared to control groups lacking additional KCl, colonies of both genotypes cultured in fresh water + 5 mM KCl displayed a significantly reduced ability to stun and capture prey, indicative of impeded nematocyst discharge. Conversely, no colonies cultured in brackish water + 5 mM KCl differed significantly from controls in ability to stun and capture prey. Colonies with a reduced ability to stun and capture prey regained their ability to do so when returned to water with no added KCl.

These data suggest that an impermanent reduction in nematocyst functionality occurs when 5 mM KCl is added in a freshwater environment. Understanding the physiology of nematocyst discharge has applications for taxonomy and species differentiation in Cordylophora spp., and these results may be used as a model for osmotic studies in other aquatic organisms.
Further Characterization of Anuran‐Like Aquaporins in the Urinary Bladder of Urodeles, Notopthalmus viridescens and Cynops pyrrhogaster
Authors: Hillyard Stanley , Shibata Yuki , Ishii Ryo , Nagai Takatoshi , Suzuki Masakazu
Published: November 2023

Recent Bayesian analysis of tetrapod aquaporin gene clusters by Finn et al (PLOS1, November 26, 2014) suggests anuran‐specific aquaporins that mediate water transport across the ventral skin (AQPa2S) and urinary bladder (AQPa2U) are more closely related to AQP6 found in mammals and reptiles. Furthermore, AQP6/a2 aquaporins are syntenic with AQP5. We used RT‐PCR to show both AQP6/a2U and AQP5 homologs are expressed in the urinary bladder of the newts, Notophthalmus viridescens (nv) and Cynops pyrrhogaster (cp). Full length amino acid sequences showed 72% and 71% identity between AQP6/a2U of nv and anuran species Hyla japonica and Xenopus tropicalis (xt). Amino acid identity of AQP5 between nv and cp was 95% while identity between both urodeles and xt was 69%–70%.

Consensus phosphorylation sites for PKA were identified for AQP6/a2U and AQP5 for both nv and cp. AQP5 could be immunolocalized in the apical membrane of bladder epithelial cells in rehydrating newts. These results indicate an absorptive role for AQP5 in the urodele urinary bladder and suggest that this role may originate in the ancestor of urodeles and anurans.

Support or Funding Information

Supported by a Grant‐in‐Aid for Scientific Research (26440165) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Evolution of the Cytochrome c Oxidase Subunit 4 Paralogs
Authors: Moyes Christopher D , Porplycia Danielle , McDonald Jared
Published: November 2023

The largest subunit of cytochrome c oxidase exists as two paralogs in most vertebrates: a constitutive COX4‐1 and hypoxia‐inducible COX4‐2. In primates and rodents, COX4‐2 is a hypoxia‐responsive gene encoding a protein with its ATP‐binding site disrupted by a disulfide bridge, precluding COX from the allosteric regulation that is exerted through the this site in COX4‐1. In an effort to understand the evolution of the gene, we examined role of the paralogs in fish. COX4‐2 of fish differs from mammals in three respects. First, the fish COX4‐2 gene does not appear to be hypoxia responsive at either the mRNA or protein level. This was shown in mammalian cells transfected with fish promoter constructs, and hypoxia treatments of cultured fish cells and whole animals. We used fish species from multiple lineages and differing in hypoxia tolerance, exposed to different degrees and durations of low oxygen. Second, the fish COX4‐2 protein lacks the structural features (paired CYS residues) that distinguish COX4‐2 from COX4‐1 in mammals. This pair of CYS residues in COX4‐2 protein appears only in the lineage of vertebrates that encompasses rodents and primates. Third, COX4‐2 expression patterns are quite different in fish. They show higher constitutive expression of COX4‐2 in all tissues, and demonstrate changes in COX4‐2 expression with development. Immunohistochemistry of tissues expressing both COX4 paralogs revealed that some tissues displayed both proteins in most cells, whereas other tissues showed cell‐specific patterns. In heart, individual myocytes expressed both COX4 paralogs, although select non‐myocytes expressed only COX4‐1. The paralog patterns also changed with size/age. Heart was dominated by COX4‐1 mRNA and protein in small fish but transitioned to predominately mRNA and COX4‐2 in large fish. The distinctions in COX4‐2 between mammalian models (rodents, humans) and other vertebrates (other mammalian lineages, reptiles, fish) bring into question the genetic origins of the paralog pair and its functional evolution during vertebrate diversification.

Support or Funding Information

Supported by NSERC Canada
Hypoxia Stimulates ERK via EGFR Activation in Human Pulmonary Microvascular Endothelial Cells
Authors: White Hilary A , Jin Yi , Chicoine Louis G , Chen Bernadette , Nelin Leif D
Published: November 2023

We have previously shown that arginase activity is necessary for hypoxia‐induced cellular proliferation in human pulmonary microvascular endothelial cells (hPMVEC) and that epidermal growth factor receptor (EGFR) activation is required for hypoxia‐induced arginase expression. We hypothesized that hypoxia‐induced EGFR activation leads to phosphorylation of extracellular signal‐regulated kinase (ERK) leading to increased arginase II expression in hPMVEC. Cells were incubated in either normoxia (21% O2, 5% CO2) or hypoxia (1% O2, 5% CO2) for 1 or 24 hours and western blotting was performed for EGFR, Arginase II, phospho‐ERK (pERK), and total ERK. We found that hPMVEC incubated in hypoxia had greater EGFR and arginase II protein levels than did normoxic cells. Hypoxia resulted in greater levels of pERK protein than in normoxic cells. To examine the role of EGFR in these hypoxia‐induced changes, hPMVEC were transfected with a siRNA against EGFR (siEGFR) or a scramble siRNA (scramble), and then incubated in hypoxia for 1 or 24 hours. Cells incubated in hypoxia and treated with siEGFR had significantly lower arginase II and pERK protein levels than did hypoxic cells treated with scramble. To determine the effect of EGFR silencing on viable cell numbers, hPMVEC were transfected with siEGFR or scramble and equal numbers of cells were plated in each well of a 6‐well plate, placed in hypoxia and 48 hours later viable cell numbers counted using trypan blue exclusion. Silencing of EGFR significantly reduced the number of viable cells seen in hypoxia. In another set of experiments, hPMVEC were treated with either vehicle, or a small molecule inhibitor of either EGFR (AG1478) or the ERK pathway (U0126) and placed in hypoxia for 1 hour. Cells were harvested for western blot analysis of pERK and total ERK. Pharmacologic inhibition of EGFR significantly attenuated hypoxia‐induced pERK protein levels, while as expected U0126 completely prevented pERK expression. Equal numbers of hPMVEC were plated in each well of a 6‐well plate and were treated with either vehicle, AG1478, or U0126, placed in hypoxia and 48 hours later viable cell numbers counted using trypan blue exclusion. Both AG1478 and U0126 significantly attenuated the hypoxia‐induced increase in viable hPMVEC numbers. Our findings suggest that hypoxic activation of EGFR results in phosphorylation of ERK, which is required for hypoxic‐induction of arginase II and cellular proliferation. We speculate that the EGFR‐ERK‐Arginase II pathway may be a potential therapeutic target to prevent or reverse the vascular remodeling seen in pulmonary hypertension associated with hypoxia.
Pulmonary Endothelial Heterogeneity in Response to Infection‐induced Cytotoxic Supernatant
Authors: Morrow Kyle Adam , Balczon Ron , Frank Dara W , Stevens Troy Adam
Published: November 2023

Here, we tested the hypothesis that Pseudomonas aeruginosa type 3 secretion system effectors ExoY and ExoU induce release of endothelial‐derived cytotoxic factors that have differential cytotoxic effects when applied to pulmonary endothelium. Pulmonary microvascular endothelial cells (PMVECs) were grown 12–24 hours post‐confluence and inoculated at a multiplicity of infection of 20:1 with P. aeruginosa possessing either ExoY (ExoY+) or ExoU (PA103). Bacterial delivery of ExoY and ExoU induced time‐dependent PMVEC gap formation that was paralleled by release of endothelial‐derived proteins, including oligomeric tau, into the extracellular space. Supernatant was collected, centrifuged for 10 minutes at 4500 × g, and filter‐sterilized. Transfer of filtered supernatant onto confluent naïve PMVECs induced apparent inter‐endothelial gap formation and destabilized pre‐formed endothelial networks in Matrigel. When filtered supernatant was transferred onto naïve pulmonary artery endothelial cells, there was a marked decrease in the cytotoxic effects when compared with PMVECs. Thus, we provide the first evidence for pulmonary endothelial heterogeneity when faced with potential disease propagation due to infection‐induced cytotoxic supernatant.

Support or Funding Information

This work was supported by American Heart Association postdoctoral fellowship grant 14POST18080004 (K.A.M.) and National Institutes of Health grants HL‐60024 and HL‐66299 (T.S. and R.B.)
Mechanisms involved in the antioxidant properties of azithromycin in lung epithelial cells stimulated with cigarette smoke extract
Authors: Cuevas Santiago , Yang Yu , Armando Ines , Jose Pedro A
Published: November 2023

Chronic Obstructive Pulmonary Disease (COPD) is a leading cause of mortality world‐wide. Macrolide antibiotics, e.g. azithromycin has antioxidant and anti‐inflammatory properties and decreases the frequency of acute exacerbations of chronic obstructive pulmonary disease (COPD). The aim of this study was to identify the molecular mechanisms whereby azithromycin decreases oxidative stress and inflammation. We have reported that azithromycin decreased intracellular Nrf2 protein level (−55±6%) and downstream phase II detoxifying enzyme NQO1 (−45±4%), reactive oxygen species (ROS) production (−1.6‐fold), and pro‐inflammatory cytokine interleukin 8 (IL‐8) secretion (−2.1‐fold) in A549 cells (human airway epithelial cells) exposed to cigarette smoke extract (CSE) (200 mg/ml, 24 h); new data indicate that histone deacetylase 2 (HDAC2) expression is also decreased (−41±4%). Pretreatment of A549 cells with azithromycin (0.5μg/ml) suppressed ROS production (−29±4%) and IL‐8 (−45±6%) induced by CSE and decreased G protein‐coupled receptor kinase 4 (GRK4) expression (−33%). Furthermore, azithromycin promoted Nrf2 nuclear translocation (270±24%) catalyzing the reduction of hyper‐oxidized peroxiredoxins (Prx) (−70±3%) leading to reduction of oxidative stress. H2O2 (200 mM/24 h) increased GRK4 (191±4%) and decreased, HDAC2 (−29±4%) expressions suggesting that oxidative stress can regulate these proteins. GRK4 co‐localize with surfactant protein A (SP‐A) (marker for alveolar epithelium type II) in human lung tissue and GRK4 and HDAC2 colocalize in human bronchus. Sestrin2, an antioxidant enzyme, was increased after treatment with azithromycin in a time and concentration dependent manner. Knockdown of Nrf2 resulted in suppression of azithromycin‐induced sestrin2 expression (−35±3%). Silencing sestrin2 abolished azithromycin‐induced decrease in peroxiredoxins hyper‐oxidation and partially attenuated the inhibitory effect of azithromycin on ROS production and IL‐8 expression (by 26% and 41%, respectively). Our results suggest that the antioxidant and anti‐inflammatory effects of azithromycin in COPD may be, in part, mediated by induction of sestrin2 via Nrf2, involving the GRK4, and HDAC2 pathways.
Investigation of the Cardiotonic Steroids, Marinobufagenin and Resibufogenin, in the Acute Respiratory Distress Syndrome
Authors: Abbas Mir Mohammad Kamran , Chen Qingzheng , Das Aayudh , Oliver Joel , Jiang Weiwu , Moorthy Bhagavatula , Patel Bela , Morin Kristi , Puschett Jules
Published: November 2023

The bufodienolides are a group of “cardiac glycosides” also termed the “cardiotonic steroids”, which are produced largely in the adrenal gland, but also in the placenta, as well as, perhaps, in the brain. They circulate in the blood and are excreted into the urine. They were originally discovered in the skin and the venom of the common toad (Bufo marinus), hence their appellation. Their major action is that of the inhibition of the ubiquitous enzyme, Na+/K+ ATPase. Acute respiratory distress syndrome (ARDS) is a life‐threatening disorder with a prevalence rate of 64 cases per 100,000 person‐years and a mortality rate of 40–52%. It is most often encountered in severely ill patients with a background of sepsis, trauma, complicated severe pneumonia and burns. Aside from improvements in ventilator therapy, there have occurred no new therapeutic strategies that improve survival. In an animal model of ARDS involving the production of the disease by exposing the animals (rats) to > 95% oxygen for 48–60 hours, serum levels of marinobufagenin (MBG) rose to 99.7 +/− 17.5 (SE) pg/ml from those determined in sham rats (mean: 25.7 +/− 2.3 SE pg/ml) (p<0.02). One hour after the hyperoxia period was completed, the intraperitoneal administration of 160 ug of resibufagenin (RBG), an antagonist of MBG, in a third group of animals, resulted in a decline of mean serum MBG to 17.2 +/− 2.7 pg/ml (p< 0.001). The mean values for MBG of the sham and hyperoxia + RBG groups differed (p= 0.003). In 18 patients with ARDS, recruited from the ICU of the Memorial‐Hermann Hospital in Houston, TX, the mean urinary excretion level of MBG/mg creatinine was 760 +/− 233 pg/mg creatinine compared to that obtained in 19 control patients admitted to the ICU for other indications was 169 +/− 74 pg/mg creatinine (p= 0.01).

We conclude that: 1) Elevated levels of MBG serve to diagnose ARDS and 2) RBG may prove to provide “rescue” therapy for this disorder.

Support or Funding Information

Jules B. Puschett, M.D. Research Endowment Fund
Chronic Intermittent Hypoxia Suppresses Adult Neurogenesis And Disrupts Synaptic Plasticity In The Dentate Gyrus Of The Hippocampus Through a Pro‐Oxidant State
Authors: Pagan Chelsea M , Khuu Maggie A , Christakis Alexander Z , Ramirez Jan‐Marino , Garcia Alfredo J
Published: November 2023

When sleep disordered breathing, such as obstructive sleep apnea (OSA), is left untreated, individuals will suffer from repeated intermittent bouts of hypoxia during sleep, leading to both cognitive decline and emotional dysregulation. The goal of our study was to determine how chronic intermittent hypoxia (CIH), an established model of OSA, affects neurogenesis and synaptic plasticity in dentate gyrus (DG) of the hippocampus, an integral site for learning and memory. Adult mice were exposed to 30 days of: (1) normoxia, (2) CIH, or (3) CIH with daily injections of the cell‐permeable antioxidant, MnTMPyP. Adult neurogenesis was investigated using immunohistochemistry on sections of fixative‐perfused DG slices. While staining for Ki‐67, a marker of proliferation, revealed that CIH stimulated mitotic division in the subgranular zone (p=0.047), the number of doublecortin (DCX) positive newly‐born granule neurons was smaller following CIH (p=0.02). This decrease in neurogenesis correlated with weakened synaptic plasticity, as observed by significantly reduced long‐term potentiation (LTP) following high‐frequency stimulation (HFS) of the perforant pathway in ex vivo DG slices (p=0.023 at 60mins post‐HFS). Treatment with MnTMPyP during CIH restored the strength of LTP. Moreover, ongoing work suggests that MnTMPyP protects neurogenesis during CIH, as the number of DCX positive cells remains similar to normoxia‐exposed controls. We conclude that, unlike other conditions associated with disturbed oxygen homeostasis, CIH differentially influences adult neurogenesis and impairs synaptic plasticity in the DG through a mechanism involving a pro‐oxidant state. Our observations in the hippocampus may be integral to understanding the mechanistic basis of cognitive decline and emotional dysregulation in patients suffering from OSA.
S‐Nitrosocysteine Alters Outward Potassium Current in Isolated Glomus Cells of Rat Carotid Body under Normoxic and Hypoxic Conditions
Authors: Getsy Paulina M. , Coffee Gregory A. , Sawczak Victoria E. , Pye Richard L. , Zaman Khalequz , Lewis Stephen J.
Published: November 2023

Glomus (type I) cells in the carotid body (CB) respond to low blood oxygen (O2) by depolarizing, opening voltage‐gated calcium channels, and releasing excitatory transmitters. These neurotransmitters activate chemosensory afferent terminals of the carotid sinus nerve (CSN), which stimulate the brainstem to increase ventilation. Our lab shows that circulating blood levels of S‐Nitrosocysteine (SNO‐L‐CYS) increase in response to hypoxia in the rat. Our objective was to determine whether SNO‐L‐CYS acts directly on the rat glomus cell to modulate its response to hypoxia. To test this we isolated glomus cells from postnatal (P) P11–P14 male and female rat pups and plated them on poly‐D‐Lysine coated coverslips. After a 2–3 hour incubation period at 37°C, 5% CO2, whole‐cell perforated patch clamp recordings were performed to characterize glomus cell current‐voltage relationship in response to 1μM SNO‐L‐CYS under normoxic (21% O2), and hypoxic (5–8% O2) conditions. Our results show that at 21% O2, 1 μM SNO‐L‐CYS increases the maximum outward steady‐state potassium current (n = 3). Under hypoxic conditions, we discovered two populations of glomus cells; (1) those whose steady‐state maximum potassium current was inhibited by hypoxia and further inhibited after administration of 1 μM SNO‐L‐CYS while in hypoxia (n=8), and (2) those whose steady‐state maximum potassium current was inhibited by hypoxia and then increased after administration of 1 μM SNO‐L‐CYS while in hypoxia (n=9). The SNO‐L‐CYS effect is washed out in recovery at 21% O2. This data suggests that under hypoxic conditions SNO‐L‐CYS has a dual‐ modulatory effect. It can act on glomus cells to further increase cell depolarization and transmitter release, leading to an increase in CSN firing. In addition, it can act on glomus cells to suppress depolarization and neurotransmitter release, thus returning the glomus cell to its resting state. Having two populations of glomus cells that respond differently to SNO‐L‐CYS may allow highly regulated carotid body firing under hypoxic conditions.

Support or Funding Information

NIH PO1‐HL1018710
Hypoxia Hits Below the Diaphragm: Effect of Acute Hypoxia on the Micturition Reflex
Authors: Catege Marianna , Solomon Irene C , Collins William F
Published: November 2023

While changes in respiratory drive and associated respiratory motor function during acute hypoxia are well documented, the effect of acute hypoxia on non‐respiratory somatic or autonomic motor systems is less well studied. The goal of the present study is to characterize the effect of acute hypoxia on lower urinary tract (LUT) function with a specific focus on reflex micturition. In urethane‐anesthetized, spontaneously breathing adult female Sprague Dawley rats, bladder intravesical pressure and external urethral sphincter (EUS) EMG activity were recorded during continuous infusion of saline into the bladder to elicit reflex micturition events. The rate of saline infusion was adjusted to achieve a baseline bladder inter‐contraction interval (ICI) of approximately 4 minutes. After 30–40 minutes of baseline recording, rats were exposed to a single episode of sustained hypoxia (10–12% O2; balance N2) with a duration of 1.5, 5 or 15 minutes followed by return to room air. The episodes of hypoxia were initiated approximately midway during bladder filling and consistently produced a premature reflex micturition event. During the 5‐ and 15‐minute episodes, the reduction in ICI continued for the duration of the hypoxia and was associated with a decrease in bladder pressure threshold (reduced intravesical pressure at the onset of active bladder contraction), particularly during 15‐minute exposures. No consistent change in EUS activity during hypoxia was observed. Following return to room air, the subsequent reflex micturition event was delayed, particularly after 5‐ and 15‐minute episodes of hypoxia, and the increase in ICI was associated with increases in both bladder pressure threshold and EUS activity during bladder filling (increased EUS guarding reflex). These data suggest that acute hypoxia alters LUT function and that both autonomic and somatic components of the micturition reflex are sensitive to hypoxia.

Support or Funding Information

Support for this project was provided by The Thomas Hartman Center for Parkinson's Research at Stony Brook University and The SUNY Brain Network of Excellence.
Becoming a couple: the developing relationship between respiratory oscillators of the American Bullfrog
Authors: Trask William M , Baghdadwala Mufaddal I , Wilson Richard JA
Published: November 2023

Many vital mammalian behaviors, including locomotion, swallowing, and breathing, are dependent upon activity of, and coupling between, neural oscillators. In the mammal, multiple respiratory oscillators—chiefly the preBötzinger complex and parafacial respiratory group—are coordinated to generate muscular outputs driving inspiratory and expiratory activity. This multiple oscillator architecture, however, is not unique to mammals. Indeed, in the bullfrog, three spatially‐distinct burst generators driving different phases of the respiratory pattern have been identified, and at least two have oscillator‐like properties. Despite extensive study of these networks in both mammals and frogs, the mechanism and development of coupling between oscillators are not fully characterized.

Over metamorphosis, the bullfrog transitions from water breathing in the tadpole to air breathing in the frog. Paralleling this shift in respiratory medium are radical changes to the underlying motor pattern produced by the brainstem, shifting from buccal ventilation in the tadpole to buccal and lung ventilation in the adult frog. To investigate the question of whether the bullfrog respiratory networks producing buccal and lung bursts ‘begin coupled’ (i.e., with tight synchronicity between oscillator rhythms upon the initial appearance of the lung pattern) or if this interaction arises over development, we employed the bullfrog isolated superfused brainstem preparation.

We recorded burst‐triggered respiratory outputs in facial and hypoglossal rootlets from isolated brainstems from early tadpole to juvenile frog. By evaluating variance in the offset between the two primary rhythms, we established coupling coefficients to characterize the oscillators’ interactions. We found evidence to suggest that, in the tadpole, the rhythms of the gill‐ventilating and nascent lung oscillators are uncoupled. Over the course of metamorphosis, these rhythms become increasingly tightly coupled, paralleling the structural and behavioral changes in the animal. Specifically, coupling appears to reach adult levels in unison with the eruption of the forelimbs. Our results indicate that the expression of the lung oscillator is effectively knit into the larger respiratory network over metamorphosis. These findings further our understanding of amphibian respiratory development and may have implications for the origins of modern air‐breathing.

Support or Funding Information

National Science and Engineering Council of Canada (NSERC) and Alberta Innovates Health Solutions (AIHS) provided funding for this study
Role of GABA and Glycine in the Development of Respiratory Rhythmogenesis in the Embryonic Zebra Finch
Authors: Stein Paxton S. , Pickett Kaci L. , Vincen‐Brown Michael A. , Pilarski Jason Q.
Published: November 2023

Primordial breathing‐related neural networks in the avian brainstem become active during the early embryonic period (day 4) but are not functional until embryonic day 12 (E12) when internal pipping occurs. At this time, birds, unlike mammals, employ active inspiration and expiration during each ventilatory cycle, but the extent to which GABA and glycine contribute to the cycle is still unknown. We aim to test the role of chloride‐mediated neurotransmission (NT) throughout this respiratory‐related development in ovo using the altricial Zebra Finch (T. guttata), which allows for unimpeded access to neural circuits during the entire embryonic period. We measured spontaneous rhythmic activity from cranial nerve IX (glossopharyngeal) between E4 and hatching (E14) in the presence and absence of the GABA/glycine antagonists bicuculline and strychnine using the in vitro brainstem spinal cord preparation. Results indicate that GABA/glycine NT is excitatory from E4 through E11. However, near E11, blocking GABA/glycine NT had little effect on the frequency of cranial nerve IX discharge but significantly increased burst area, which suggests an increase in phasic excitation of the glossopharyngeal motor nucleus.

Support or Funding Information

IDeA Network of Biomedical Research Excellence (INBRE), NIH grant P20 PR016454

NIH Academic Research Enhancement Award (AREA) Program, 1R15NS087521‐01A1
ATP Excitation of the PreBötzinger Complex and Inspiratory Neurons in vitro is Mediated in Part via a PLC/IP3‐mediated increase In [Ca2+]i
Authors: Zhang Yong , Gourine Alexander , Kasparov Sergey , Alvares Tucaaue , Funk Gregory
Published: November 2023

The ventilatory response to hypoxia comprises an initial increase in ventilation followed by a secondary depression that can be life‐threatening in premature infants. ATP released within the ventral respiratory column, including the preBötzinger Complex (preBötC, critical site for inspiratory rhythm generation), during hypoxia attenuates this depression. Astrocytes sense hypoxia via a mitochondrial mechanism that involves mitochondrial depolarization, free radical production, lipid peroxidation, phospholipase C (PLC) and inositol trisphosphate (IP3) activation followed by an increase in intracellular Ca2+ and vesicular ATP release. Our working hypothesis is that ATP released from preBötC astrocytes during hypoxia acts in an autocrine/paracrine manner to enhance gliotransmission and also excites local inspiratory neurons directly via a P2Y1 receptor (R)‐dependent mechanism to increase inspiratory frequency (freq). Our first objective was to determine if the ATP excitation of inspiratory neurons includes an astrocytic glutamate component (i.e., do astrocytes release glutamate in response to P2Y1 receptor activation?). Using 700 μm rhythmically‐active medullary slices, we obtained whole‐cell recordings from preBötC inspiratory neurons and compared neuronal ATP (5 mM) currents evoked before and after bath application of glutamate R antagonists AP5 (100 μM) and CNQX (10 μM) (in 0.5 μM TTX). AP5 and CNQX had no effect on ATP currents (n=8). Our second objective was to identify the signaling pathways through which ATP excites the preBötC network and neurons. Simultaneous application of the PLC blocker U73122 (20 μM) to the bath (20 μM) and into the preBötC (10 μM) via pressure injection shortened the duration of MRS2365 (100 μM)‐evoked frequency increase but had no effect on the peak frequency (n=9). 2‐APB (100 μM), an IP3R inhibitor, reduced the ATP‐evoked freq increase from 232.14 ± 39% to 147 ± 12% (n=5). In contrast, intracellular dialysis of U73122 (2 μM, n=6) and 2‐APB (50 μM, n=4) attenuated ATP‐evoked inward currents in inspiratory neurons by 50 ± 7% and 42 ± 17%, respectively. Bath application of U73122 had no effect on the ATP‐induced inward current (n=4), suggesting that bath‐applied U73122 does not access the neurons. Intracellular dialysis of sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) blocker thapsigargin (4 μM, n=3) also attenuated the ATP‐induced inward currents in inspiratory neurons by 29.3 ± 1.03%. These data suggest that although the excitation of preBötC inspiratory neurons by ATP does not include a glial‐derived glutamatergic component, network excitation is brought about, at least in part, via activation in inspiratory neurons of a PLC, IP3 receptor‐mediated release of Ca2+ from intracellular stores.

Support or Funding Information

Supported by CIHR, AIHS, CFI, WCHRI and ALA.