bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of Circadian CLOCK genes alters proinflammatory cytokine production in human alveolar epithelial cells
Authors: Parthasarathy Prasanna Tamarapu , Luong Tran , Lagishetty Venu , Phillips Oluwakemi , Cox Ruan , Castillo Annie , Abuelenen Toaa , Lockey Richard , Kolliputi Narasaiah
Published: June 2021

Acute lung injury (ALI) is a devastating clinical syndrome manifested by inflammation and decreased lung compliance. Cytokines such as IL‐1b, IL‐6 and IL‐8 have been implicated in the process of inflammation and in chemokine stimulation associated with ALI. Recent studies suggest that circadian clock genes are involved in inflammation disorders; however, the role of these genes in ALI is yet unknown. Therefore, we investigated the role of clock genes on proinflammatory cytokine production in human alveolar epithelial cells (A549). A549 cells were transfected with core clock genes (Bmal1, CLOCK, Cry1/2, Per1, Per2) and subsequently treated with either IL‐1b or hydrogen peroxide. Supernatants were analyzed for the production of IL‐6 and IL‐8. There was a significant decrease in IL‐8 production in cells transfected with core CLOCK genes. However, the level of IL‐6 was unchanged in these cells. Further, A549 cells treated with IL‐1b or hydrogen peroxide after Cry1 and Per2 transfection showed a significant decrease in IL‐6 and IL‐8. Our data suggest that core clock genes may be protective by ameliorating cytokine production thereby offering a potential therapeutic to minimize the clinical severity and mortality of ALI.

This work was supported by the AHA 09SDG2260957 and NIH R01 HL105932 grants to N.K.
Modeling the alveolar Ar after been squeezed by inspiring room air
Authors: Melendrez Jose Maria , Caucha Luis Jhony , Cruz Julio Cesar
Published: June 2021

The experiment of inhaling 1.9L of 79% argon in O2 was modeled previously (Cruz. Respir. Physiol. 86:1, 1991). To account for the argon inhaled, 1.501L, a large dead space, 0.425L, was necessary. Moreover, 1.9L of air was inhaled to reach TLC. Thus, the alveolar Ar that was inhaled previously was squeezed and pushed in to the end of Weibel's generations. We recently showed that the expired CO2, previously diluted in the alveoli, became more evident with the forced expiration and revealed the effect of alveolar CO2 diffusion from RV to ERV (Caucha's et al. FASEB J. 24:1063.5, 2012). Now, we modified Caucha's model to simulate the Ar expirogram, changing the number of generations assigned for convection, convection‐diffusion and diffusion in the seven parallel regions from apex to base of the lung. The Figure shows two kinds of experiments, the first one simulates the experiment of Cruz in 1991. This is depicted on the left scale (dashed curve) with a volume expired of 4.5L. The “sandwich” experiment is shown on the right scale (for clarity) with a volume expired of 6.4L. The total argon inhaled is now in the alveoli and 1.24L of Ar was exhaled. Thus, more Ar was diffused to RV to reach 0.234L in the model presented (error of 1.7%). Note that the curve shown is the one that would be obtained during expiration after the alveolar argon was diluted and squeezed by inhaling air. The alveolar plateau reached the ideal alveolar gas mixing, 0.20 [Amount of Ar/(Total Lung Capacity‐Dead space)] at 3.8L‐4.23L (horizontal dotted line). However, the slope of phase IV is negative.

Supported in part by CEIS (Centro de Enseñanza, Investigación y Servicios).
Direct demonstration that blood flow through intrapulmonary arteriovenous anastomoses worsens pulmonary gas exchange efficiency
Authors: Elliott Jonathan E. , Laurie Steven S. , Beasley Kara M. , Goodman Randall D. , Gladstone Igor M. , Hawn Jerold A. , Lovering Andrew T.
Published: June 2021

Pulmonary gas exchange efficiency is defined and quantified by the alveolar‐arterial PO2 difference (AaDO2). Sources of venous admixture (QVA/QT) that increase AaDO2 are ventilation‐perfusion (V/Q) inequality, diffusion limitation, and shunt (QS/QT). Intravenous infusion of epinephrine (EPI) increases blood flow through intrapulmonary arteriovenous anastomoses (QIPAVA) in healthy humans at rest, as detected by saline contrast echocardiography, but whether or not this is a source of shunt remains controversial. We hypothesized that QIPAVA is a source of shunt, and therefore, AaDO2 would increase during EPI infusion in healthy humans at rest when QIPAVA is increased. From baseline to max EPI infusion (320 ng/kg/min), AaDO2 increased from 4.9 to 9.4 mmHg and QVA/QT increased from 2.8 to 3.8% in healthy subjects breathing room air (n=3). We repeated these measurements in the same subjects breathing 40% O2 which increases alveolar PO2 and reduces O2 equilibration time from ~0.6 to <0.2 sec, thereby eliminating QVA/QT contributions from diffusion limitation and V/Q inequality, but still allowing for QIPAVA to occur. From baseline to max EPI infusion breathing 40% O2, AaDO2 increased from 17.3 to 23.7 mmHg and QS/QT increased from 4.4 to 5.4%. We conclude that QIPAVA is a source of shunt, because QVA/QT and QS/QT both increased by 1% during max EPI infusion. Support: American Heart Association Predoctoral Fellowship
Endothelial glycocalyx degradation predisposes for transfusion‐associated acute lung injury
Authors: Schmidt Eric P , Barthel Lea , Kelher Marguerite , Perez Mario J , Smith Lynelle P , Silliman Christopher C , Benson Alexander B , Janssen William J
Published: June 2021

Transfusion‐associated acute lung injury (TRALI), a major cause of transfusion morbidity, arises from two events: an initial alteration in host susceptibility, followed by exposure to mediators in transfused blood products. We hypothesized that pulmonary endothelial glycocalyx degradation was a predisposing event in TRALI. We created a mouse model of TRALI, administering IV lipopolysaccharide (LPS) 2h prior to the administration of bioactive lipids (arachidonic acid, lysophosphatidylcholine) identified in stored human RBC units. Enzymatic cleavage of glycocalyx heparan sulfates similarly predisposed for TRALI, suggesting a role for glycocalyx degradation in host susceptibility to injury. Glycocalyx degradation could be detected in human blood via identification of heparan degradation products, defined by a heparinase effect of >; 15% on thromboelastography r‐time. Presence of circulating heparinoids was associated with a 50% risk (2/4) of plasma transfusion‐induced lung injury in bleeding patients with liver disease; patients with no circulating heparinoids had 0% risk (0/3). Endothelial glycocalyx degradation predisposes for TRALI and can serve as a biomarker for transfusion risk. NHLBI K08HL105538.
Endostatin limits pulmonary endothelial repair via RhoA inactivation
Authors: Jian Ming‐Yuan , Creighton Judy
Published: June 2021

Endothelial repair following vascular injury involves cell proliferation and migration. Endostatin regulates cell proliferation, and is essential for maintenance of a functional vascular barrier. However, elevated endostatin levels correlate with severity of inflammatory lung damage. The source of endostatin or its role in progression of vascular disease is not known. We reasoned endostatin release by lung endothelium during an injury event contributes to pulmonary vascular damage by limiting endothelial proliferation and migration; thus slowing barrier restoration. LPS increased endostatin expression in lung tissue in vivo and in pulmonary microvascular endothelial cells (PMVECs) in vitro. LPS caused translocation of endostatin from peri‐nuclear regions to cell membranes in PMVECs consistent with release of endogenously synthesized endostatin. LPS inhibits PMVEC proliferation and wound healing, an effect exacerbated by exogenous application of endostatin. Endostatin decreased RhoA activity in PMVECs implicating the RhoA pathway in endostatin signaling.

Conclusion

inflammation induces endostatin release from endothelium sufficient to limit lung vascular repair. Supported by HL102296 and HL 110803.
HIF‐2α regulates hypoxia‐induced arginase II expression through EGFR
Authors: Cui Hongmei , Liu Yusen , Chicoine Louis G , Chen Bernadette , Nelin Leif D
Published: June 2021

To test the hypothesis that arginase II is induced in an oxygen‐dependent manner in human pulmonary microvascular endothelial cells (hPMVEC), cells were incubated in either normoxia (20% O2) or hypoxia (1% O2) for 6 or 24 h, and some of the hypoxic hPMVEC were returned to normoxia or hypoxia for an additional 24 h. The hPMVEC incubated in hypoxia for 24 h produced ~70% less NO than cells incubated in normoxia (p<0.01). Greater HIF‐2α, EGFR, and arginase II protein levels were found in hypoxic hPMVEC than in normoxic cells (p<0.05). Hypoxic hPMVEC returned to normoxia had substantially greater NO production, while arginase II, HIF‐2α, and EGFR protein levels were lower, than in hypoxic hPMVEC (p<0.05). hPMVECs were transfected with siRNA for 48 h to knockdown the expression of HIF‐2α or EGFR. We found that silencing HIF‐2α prevented hypoxia‐induced EGFR and arginase II expression. Conversely, silencing of EGFR also prevented HIF‐2α and arginase II expression, as well as downstream ERK and Akt activation. These results suggest that there is a feedback regulation loop between HIF‐2α and EGFR. Pharmacological inhibition of Akt and MEK1/2 prevented hypoxia‐induced arginase II expression. Thus, arginase II is induced by hypoxia through a pathway mediated by HIF2α, EGFR, ERK, and Akt. We speculate that arginase II regulation may represent a potential therapy for pulmonary hypertension associated with hypoxia.
Hemin‐Induced miR‐27a Reduces PPARγ Expression in Sickle Cell Disease with Pulmonary Hypertension
Authors: Kang Bum‐Yong , Park Kathy K , Tan Fang , Ghosh Samit , Sutliff Roy L. , Ofori‐Acquah Solomon F. , Hart C. Michael
Published: June 2021

The hallmark of sickle cell disease (SCD) is hemolysis, vaso‐occlusion, and oxidative stress. Pulmonary hypertension (PH) is a serious complication of SCD that causes significant morbidity and mortality. We previously demonstrated that: 1) activation of the nuclear hormone receptor, peroxisome proliferator‐activated receptor gamma (PPARγ attenuated hypoxia‐induced Nox4 and endothelin‐1 (ET‐1) expression and PH in mice and 2) that levels of Nox4 and ET‐1 were increased in the lungs of 8–10 week old SCD transgenic mice compared to controls whereas PPARγ levels were reduced. The current study further examines mechanisms of PH in SCD. Human pulmonary arterial endothelial cells (HPAECs) were treated with the hemolysis product, hemin (5 μM), for 72 h. Hemin reduced PPARγ and increased Nox4 and ET‐1 expression and HPAEC proliferation. MicroRNA‐27a (miR‐27a), which negatively regulates PPARγ, was increased in hemin‐treated HPAECs. In contrast, treating HPAECs with the PPARγ rosiglitazone (10 μM) for the final 24 h of hemin exposure attenuated increased HPAEC proliferation and miR‐27a levels. These findings suggest that hemin increases miR‐27a to reduce PPARγ and increase Nox4, ET‐1, and PAEC proliferation. These results suggest novel pathways for SCD‐PH pathogenesis and therapy. Supported by Atlanta VA Merit Review, NIH HL102167, and Emory/Childrens Healthcare CEB F16788–00.
Membrane cholesterol facilitates agonist‐induced calcium entry in pulmonary arterial endothelium
Authors: Zhang Bojun , Naik Jay , Resta Thomas , Walker Benjimen
Published: June 2021

ATP‐induced calcium entry(ATP‐CE) along with membrane cholesterol levels are reduced in pulmonary artery endothelial cells (PAECs) from rats exposed to chronic hypoxia (CH; 4 wk, PB = 380 torr). Furthermore, supplementation of membrane cholesterol restores ATP‐CE in PAECs from CH rats without affecting controls. To further elucidate the role of membrane cholesterol in agonist‐induced calcium entry, we studied on PAECs from control animals. We hypothesized that PAEC membrane cholesterol facilitates agonist‐induced calcium entry. To test this hypothesis, we administered the cholesterol depleting agent methyl‐β cyclodextrin (MβCD) or vehicle to isolated PAEC sheets from control rats and assessed ATP‐CE with fura‐2 fluorescence. Calcium entry was reduced in PAECs treated with MβCD compared to controls. Treatment with epicholesterol (epimeric form of cholesterol) similarly decreased ATP‐CE. In contrast, cholesterol‐saturated MβCD had no effect on ATP‐CE. We conclude that membrane cholesterol facilitates agonist‐induced calcium entry in PAECs and that the reduced influx seen after CH is possibly due to loss of this key regulator. Supported by NIH grant HL95640.
Long Isoform of Myosin Light Chain Kinase Interacts with Calcium Release‐Activated Calcium Channel Constituents to Induce an Amplified and Protracted Increase in Intracellular Calcium
Authors: Tauseef Mohammad , Kini Vidisha , Sharma Tiffany , Knezevic Nebojsa , Thennes Tracy , Vogel Stephen , Gallagher Patricia , Malik Asrar , Mehta Dolly
Published: June 2021

Stromal interaction molecule 1 (STIM1) induces calcium (Ca2+) entry by activating transient receptor potential canonical (TRPC) channel‐mediated store‐operated calcium (SOC) entry or ORAI1 mediated Ca2+ release‐activated Ca2+(CRAC) entry. However, mechanisms regulating STIM1 interaction with ORAI1/TRPC1 remains unclear. Myosin light chain kinase (MLCK)‐210, a Ca2+‐calmodulin dependent long isoform of MLCK, contains domains at N‐terminus which mediates protein‐protein interaction. We tested the hypothesis that MLCK through its N‐terminus interacts with STIM1, ORAI1 and TRPC1 leading to amplified and protracted increase in Ca2+ entry. We show that thapsigargin, which activates SOC as well as CRAC channels, induce markedly attenuated increase in Ca2+ entry in MLCK‐210−/− endothelial cells (ECs). Restoring N‐terminus but not C‐terminus of MLCK‐210 rescued Ca2+ entry in MLCK‐210−/− ECs. Over expression of N‐MLCK‐210 mutant in wild type ECs rapidly enhanced Ca2+ entry which had a steep slope quite distinct than vector expressing ECs. Interestingly, MLCK‐210 interacted with ORAI1, STIM1 and TRPC1 under basal condition and the interaction further increased following thapsigargin stimulation of ECs. These observations suggest that MLCK‐210 may facilitate STIM1 interaction with ORAI1 and TRPC1 to amplify and protract Ca2+entry in ECs
H202 regulates lung ENaC via ubiquitin‐like protein Nedd8 in alveolar type 1 and type 2 cells.
Authors: Johnson Nicholle M. , Downs Charles A. , Kreiner Lisa H. , Helms My N.
Published: June 2021

Proteolytic degradation of epithelial sodium channels (ENaC) assists in regulating net salt and water balance in lung epithelia. Classical ubiquitination of ENaC involves Nedd 4–2; however, less attention has been given to the role of ubiquitin‐like protein Nedd8. We tested the hypothesis that Nedd8 and its associated E2 carrier protein (Ubc12) and E3‐SCF ligase, plays a key role in regulating the expression of ENaC in alveolar type 1 (T1) and type 2 (T2) cells. Using single channel patch clamp analysis in the cell attached configuration, we exposed alveolar T2 cells to 250 μM H2O2 and observed that channel open probability increased by 35 min exposure (p <0.05) with a significant increase in α‐ENaC protein at 60 and 120 minutes (p<0.05). Patch clamp analysis of T1 and T2 cells treated with MLN 4924, a specific Nedd8 activating enzyme inhibitor, showed a significant increase in ENaC activity. Cycloheximide chase assays performed +/− MLN 4924 showed a significant increase in α‐ENaC in MLN4924 treated cells compared to cycloheximide alone at 60 and 120 minutes (p<0.05). MLN 4924 treated mice showed accelerated rates of alveolar fluid clearance, confirming our electrophysiological measurements and supporting a significant role for Nedd8 in the ubiquitination of α‐ENaC. Research was supported by R00HL09226 awarded to MNH; Children's Center for Development in Lung Biology Grant awarded to MNH
Domain specific regulation of Src kinase by Na/K ATPase alpha1 subunit
Authors: Banerjee Moumita , Duan Qiming , Ye Qiqi , Li Zhichuan , Xie Zijian
Published: June 2021

Invitro binding assay suggests that the α1 subunit of Na/K ATPase interacts with Src via two separate domain interactions‐ the nucleotide binding domain and second cytosolic domain (CD2) binds kinase and SH2 domain of Src respectively. The aim of this work was to assess the functionality of the putative CD2‐SH2 interaction in cellular signal transduction. LLC‐PK1 cells were transfected with either YFP or YFP‐CD2, several clones were generated and characterized. We found that expression of YFP‐CD2 significantly increased basal Src activity and consequently overall protein phosphorylation. YFP‐CD2 disrupted the interaction between Na/K ATPase and Src thereby inhibiting ouabain mediated signal transduction in cells. It also effectively attenuated other Src signaling pathways like cell attachment induced FAK activation. Consistently it inhibited cell spreading and proliferation, both of which require Src signaling. Finally, the C terminal half of CD2 was sufficient in eliciting the inhibitory effects of CD2 on Src kinase. These observations therefore support our notion that CD2 of α1 subunit interacts with Src SH2 domain and is critical for formation of Na/K ATPase/Src receptor complex for ouabain mediated cell signaling. Thus, CD2 acting as a dominant negative mutant can block Src activation dependent signaling pathways via a mechanism different from general Src inhibitors. NIH grant HL36573, GM78565
Na/K‐ATPase in Bone‐Marrow Derived Stromal Cells
Authors: Sayed Moustafa Helmy , Liu Jiang , Xie Zijian , Shapiro Joseph I , Tian Jiang
Published: June 2021

Bone‐marrow derived stromal cells (BMSCs) have the capacity to differentiate multi‐directionally. We have demonstrated that the signaling function of Na/K‐ATPase plays an important role in regulating cell survival and cell growth. This study is to test if Na/K‐ATPase and its signaling functions affect BMSCs survival and differentiation capacity. BMSCs isolated from Sprauge‐Dawley male rats were cultured in α‐MEM containing 15% FBS. The purified third generation of BMSCs was treated with 10nM or 1μM marinobufagenin (MBG, a cardiotonic steroid) for 72h then the cells were induced to differentiate into adipocytes or osteoblasts using 1μM Rosiglitazone or 10mM β‐glycerophosphate, respectively. The results illustrated that MBG treatment activates the Na/K‐ATPase signaling pathway and promoted Rosiglitazone‐induced adipogenesis. In addition, MBG treatment resulted in a trend of decreased osteogenesis induced by β‐glycerophosphate, but not in a statistically significant manner. To evaluate the mechanism behind this phenomenon, BMSCs were pretreated with 1μM PP2 (a Src inhibitor) for 30 minutes, then cells were treated with 10nM and 1μM MBG for 72 hours before the induction of adipogenesis. This combined treatment decreased adipogenesis when compared to cells treated with MBG alone. These results suggest Na/K‐ATPase and its signaling functions are involved in the regulation of BMSCs differentiation. Supported by NIH HL‐105649
Cisplatin‐induced cell death in MCF‐7 breast cancer cells: Roles of ΔNErbB2 and pH regulatory ion transporters NHE1 and NBCn1
Authors: Sigurđsson Haraldur H. , Olesen Christina W. , Dybboe Rie , Lauritzen Gitte , Pedersen Stine Falsig
Published: June 2021

We have shown that expression of a truncated constitutively active ErbB2 receptor (ΔNErbB2), which is associated with poor prognosis in breast cancer, greatly increased net acid extrusion in MCF‐7 breast cancer cells in a manner mediated by the Na+/H+ exchanger NHE1 and the Na+,HCO3− cotransporter NBCn1. Here, we explore the roles of NHE1 and NBCn1 in cisplatin‐induced cancer cell death. In both vector‐ and ΔNErbB2 cells, cisplatin treatment (25 μM, 18 h) induced cleavage of caspase‐9,‐7, PARP, and fodrin (a calpain substrate), and cysteine cathepsin release, all in a manner slightly augmented by NHE1 inhibition. Expression of the anti‐apoptotic Bcl‐2 protein was markedly reduced in ΔNErbB2‐ compared to vector cells. Cisplatin treatment increased p53‐ and PUMA levels in both cell types, and reduced the number of autophagic vesicles and increased p62 levels, most markedly in ΔNErbB2 cells. This reduction of autophagic flux was not significantly affected by inhibition of NHE1 or NBCn1. Finally, cisplatin treatment elicited marked perinuclear accumulation of NBCn1, but not of NHE1, and reduced the protein levels of both full length‐ and ΔN‐ErbB2. In conclusion, cisplatin treatment of MCF‐7 cells activates several death mechanisms, some of which are further exacerbated by NHE1 inhibition in agreement with previous findings that NHE1 inhibition sensitizes MCF‐7‐ΔNErbB2 cells to cisplatin‐induced death.

Funding: Danish Cancer Society, Danish Research Council
Peptide affinity chromatography identifies proteins that bind to N‐terminus of NFAT5
Authors: Izumi Yuichiro , Jinxi Li , Burg Mauice B. , Ferraris Joan D.
Published: June 2021

Nuclear Factor of Activated T‐Cells 5 (NFAT5) induces transcription of osmoprotective genes in response to hypertonicity. NFAT5 is associated with many other proteins, only a few of which have been identified. In order to identify additional proteins that interact with NFAT5, we performed peptide affinity chromatography. Biotin‐tagged peptides were synthesized, each containing 20 amino acids from a region in the N‐terminus of NFAT5 predicted to be intrinsically disordered. HEK293 cells were grown in light, medium, or heavy SILAC medium, then incubated at 200, 300, or 500 mOsm (NaCl varied), respectively, for 30 min. Cytoplasmic or nuclear extracts from the different osmolalities were combined and incubated with peptides and streptavidin‐coated magnetic beads to isolate proteins associated with the peptides. Proteins eluted from the beads were analyzed on an Eksigent nanoLC Ultra coupled with a Thermo LTQ‐Orbitrap Velos mass spectrometer. LC‐MS/MS files were searched via Proteome Discoverer (PD) against Sprot human database for peptide and protein identification. Numerous proteins were reproducibly identified in biological replicates associated with peptide baits 131 – 150, 161 – 180, 191 – 210 and 206 – 225 in nuclear and/or cytoplasmic fractions. Peptide affinity chromatography revealed proteins that are associated with NFAT5 and that presumably regulate it in response to hypertonicity.
Ion regulation in eurythermic teleost fish Fundulus heteroclitus: Cold shock vs. cold acclimation
Authors: Marshall William S , Barnes Kaitlyn R , Malone Alicia M , Buhariwalla Hannah EC , Osmond Emily M
Published: June 2021

Killifish overwinter (0–5°C) by decreasing activity and metabolic rate and yet they retain ability to ion/osmoregulate. Fish were acclimated to seawater (32 ppt) at 5°C and 20°C (8+ weeks); cold shock of 20°C opercular epithelia in vitro reversibly decreased Clsecretion by 90% (as short‐circuit current, SCC) and membranes failed to respond to hypotonic shock, IBMX/db‐cAMP (0.1 mM) or isoproterenol (1.0 μM). Opercular epithelia from cold‐acclimated fish also had low transport rates, but were able to respond normally, i.e. rapid inhibition of SCC by hypotonic shock and SCC stimulation by IBMX/db‐cAMP and isoproterenol. Cold acclimation caused homeoviscous adaptive changes, increased fatty acid unsaturation and increased short chain (e.g. reduced C18:0 and increased C14:0, C18:1) to maintain membrane fluidity. Cooling in vitro to 1°C induced a sharp bend in Arrhenius plots below 8°C for warm (Q10 = 6.9) and cold (Q10 = 4.2) acclimated fish, but these nonlinearities were markedly reduced (to Q10 = 2.9) by anaerobiosis and cyanide (0.5 mM), indicating mitochondrial inner membrane ATP production as source of the transition. In the cold, Fundulus markedly reduce ion transport rates but maintain regulatory control of ion transport through homeoviscous adaptation. Supported by NSERC, CFI and UCR.
ATP release from exocrine pancreatic cells
Authors: Kowal Justyna Magdalena , Haanes Kristian Agmund , Novak Ivana
Published: June 2021

Extracellular ATP plays important roles as a signaling molecule between various cells in our body. In exocrine pancreas ATP is secreted from acinar zymogen granules into the pancreatic duct (Haanes & Novak 2010 Biochem J 429:303–311). In ducts, this ATP, possibly together with ATP released locally, can act on P2X/P2Y receptors and thereby regulate pancreatic ductal secretion (Wang & Novak 2012 Pancreas Epub. PMID:22982819). Our aim was to determine physiological and pathophysiological stimuli that release ATP in pancreatic ducts. Luciferase/luciferin bioluminescence assay was used to determine concentrations of ATP released from human pancreatic duct cells (Capan‐1) in response to various stimulants. We observed fast and long‐lasting ATP release in Capan‐1 cells in response to hypotonic and mechanical stimuli. ATP release observed with addition of UTP (10 μM) and ionomycin (5 μM) was slower. Interestingly, the bile acid chenodeoxycholate (0.5 mM) induced high and fast ATP release (0.96 ± 0.19 μM/106 cells/ml). Together our data show that there are various stimuli causing ATP release with different kinetics and this may indicate different releasing mechanisms. In pancreatic ducts, ATP release might be an important co‐stimulatory and protective mechanism: it stimulates fluid secretion, possibly washes out intra‐luminal obstructions or bile acids that might enter pancreatic ducts. S: Lundbeck Fdn, DNSRC
Decreased calcium mobilisation and the lack of secretion in equine sweat gland cells
Authors: Robertson Jennifer , Ko Wing‐Hung , Bovell Douglas
Published: June 2021

Sweat secretion helps maintain skin surface ecosystems and allows humans and the Equidae to thermoregulate. A key component in sweat secretion is an increase in intracellular Ca2+ ([Ca2+])i from internal and external sources. Regulation of Ca2+ entry across the plasma membrane is initiated by emptying of the intracellular Ca2+stores and through a process known as store–operated Ca2+ entry (SOCE) via STIM and Orai proteins.

We studied the role of SOCE in horse sweat gland cells from control animals and those that had developed anhidrosis, an inability to produce sweat.

Western blot analysis and PCR confirmed the presence of STIM and Orai proteins in cell lines derived from control and anhidrotic animals. However, the results indicated a reduction in STIM1 mRNA and protein expression, as well as a decrease in the localisation of STIM1 in the ER of anhidrotic cells compared to control cells. The functionality of the SOCE pathway in both cell lines, in response to agonist stimulation was investigated using (FURA‐2AM) calcium imaging. Increases in [Ca2+]i could be abolished in the presence of pharmacological blockers of SOCE and that anhidrotic cells showed a significant decrease in the [Ca2+]i FURA‐2 ratio in response to agonists when compared to control cells.

The results suggest that a reduction of STIM1 expression in the SOCE pathway may play a role in anhidrosis in horses.

Support provided by the Caledonian University.
Purinergic signaling integrates local excitation and global inhibition signals that regulate neutrophil chemotaxis
Authors: Bao Yi , Chen Yu , Ledderose Carola , Li Linglin , Junger Wolfgang
Published: June 2021

Neutrophil chemotaxis requires local excitatory signals at the front and inhibitory signals at the back of cells. The mechanisms that link these signals are unknown. We previously showed that chemoattractants induce ATP release and excitatory purinergic feedback mechanisms at the front. Here we show that pannexin‐1 (panx1) is responsible for ATP release and that chemotaxis requires translocation of panx1 to the front and A2a adenosine receptors (A2aR) to the back. Panx1 provides both, ATP and adenosine, to elicit the excitatory and inhibitory signals at the front and back of cells. Inhibition of panx1 with carbenoxolone (CBX) completely abolished chemotaxis, while inhibition of A2aR with the antagonist CSC reduced migration speed (Fig. 1). Our findings demonstrate that panx1 is the molecular link that integrates the excitatory and inhibitory purinergic feedback mechanisms that regulate chemotaxis at the front and back of cells.

This work was funded by grants from the National Institutes of Health and the Congressionally Directed Medical Research Program.
Actin association and stimulus‐specific activation of ERK1/2 in smooth muscle cells are regulated at the level of ERK1/2 scaffolds
Authors: Vetterkind Susanne , Poythress Ransom , Lin Qian Qian , Morgan Kathleen G
Published: June 2021

In smooth muscle, the extracellular signal regulated kinases 1 and 2 (ERK1/2) contextually regulate contraction or proliferation. Here, we test the hypothesis that ERK1/2 scaffolds regulate the intracellular localization and consequently, stimulus‐specific outcomes of ERK1/2 activation. SiRNA against the ERK1/2 scaffold IQGAP1 impairs phorbol ester induced ERK1/2 phosphorylation, but has no effect on basal or serum induced ERK1/2 phosphorylation in A7r5 smooth muscle cells. IP experiments show that ERK1/2 associates with both, IQGAP1 and kinase suppressor of ras‐1 under unstimulated conditions and dissociates upon serum stimulation. However, only IQGAP1 remains associated with ERK1/2 after phorbol ester stimulation. Immunofluorescence imaging and proximity ligation assays show that phorbol ester induced actin binding of phospho‐ERK1/2 is reduced after siRNA knock down of either IQGAP1 or caveolin‐1 (CaV). Interestingly, actin binding of total ERK1/2 is impaired by IQGAP1 siRNA, but not CaV siRNA. Simultaneous knock down of IQGAP1 and CaV reduces phorbol ester induced phospho‐ ERK1/2 to the same degree as IQGAP1 or CaV knock down alone, indicating that these two scaffolds act in the same ERK1/2 activation pathway. In summary, our results show that ERK1/2 scaffolds control distinct ERK1/2 pools and modulate the outcome of ERK1/2 activation in a stimulus‐specific manner. Support: HL080003, HL086655.
Direct interaction with the Na+/H+ exchanger NHE1 regulates ERK1/2 activity
Authors: Johansen Jane , Hendus‐Altenburger Ruth , la Cour Jonas , Kragelund Birthe B. , Pedersen Stine F.
Published: June 2021

Increased activity of extracellular signal regulated kinase (ERK)1/2 plays a central role in cancer pathology. More recently, the Na+/H+ exchanger NHE1 has also been assigned important roles in cancer development and chemotherapy resistance. Using a combination of fluorescence spectroscopy, NMR analysis, and chemical crosslinking experiments we characterized the interaction of human (h) NHE1 C‐terminal cytoplasmic distal tail (NHE1Cdt) and ERK2 in vitro, and found that NHE1Cdt and ERK2 physically interact with low affinity and with exploitation of D‐domains as identified from distinct changes in their chemical shifts upon titration with ERK2. The hNHE1Cdt contains two predicted D‐domains ‐ a typical feature of ERK‐interacting proteins ‐ located around amino acids 715 (D1) and 740 (D2), respectively. The functional effects of NHE1‐ERK interaction were evaluated by mutating the D‐domains in full‐length hNHE1 (D1D2 mutant) and expressing wt and mutant NHE1 in epithelial cells lacking endogenous NHE1. Wt‐ and D1D2 mutant hNHE1 appeared to target normally to the plasma membrane. D1D2 mutation abolished EGF‐induced ERK activation, and both D domains appeared to be required for full ERK activity. In contrast, net acid extrusion capacity after an acid load was similar in cells expressing D1D2 mutant‐ and wt hNHE1. In conclusion, ERK physically interacts with hNHE1 and this appears to regulate ERK activity.

Funding: Danish Research Council
The BK channel in the renal adaptation to chronic metabolic acidosis (CMA) in cortical collecting duct (CCD)
Authors: Schreck Carlos M , Zavilowitz Beth , Satlin Lisa M
Published: June 2021

The density of BK channels in CCD intercalated cells (ICs) exceeds that in principal cells. We hypothesized that this channel is upregulated in acid secreting α‐ICs during CMA to recycle K for a luminal H/K‐ATPase. H pump activity was measured in α‐ICs in BCECF‐loaded microperfused CCDs from CMA rabbits (75 mM NH4Cl in drinking water × 5–7 d) as the rate (pH U/min) of Na‐independent pHi recovery from an acute NH4Cl prepulse in the absence (H‐ATPase) and then presence (H/K‐ATPase) of luminal (L) K, in the absence (control) or presence of iberiotoxin (IBX), a BK channel inhibitor. The effect of IBX on pHi was also studied in the presence of bath EIPA and L bafilomycin (Baf) to inhibit Na/H exchange and the H‐ATPase, respectively.

Nadir pHi
H‐ATPase activity
pHi
H/K‐ATPase activity

Control (n=7)
6.64 ± 0.05
0.057 ± 0.030
6.89 ± 0.08
0.009 ± 0.011

L IBX (n=4)
6.68 ± 0.05
0.043 ± 0.040
6.87 ± 0.10
0.000 ± 0.008

In 5 CCDs, pHi fell from 7.36 ± 0.10 to 7.16 ± 0.06 with EIPA (p<0.05) but was further unchanged by L Baf or IBX. The findings that IBX did not significantly alter (i) H‐ or H/K‐ATPase‐mediated recovery from an acute acid load or (ii) pHi in CCDs from CMA animals suggests that the channel does not participate in the adaptation of α‐ICs to CMA.
Regulation of V‐ATPase in mouse hippocampal neurons following metabolic‐induced‐ and activity‐dependent acid‐base changes
Authors: Roussa Eleni , Speer Jan Manuel , Oehlke Oliver
Published: June 2021

Changes in extracellular pH (pHo) are common events associated with both physiological conditions and pathological states in brain function. Neuronal activity is associated with extracellular alkalinization and a rapid intracellular acidification whereas significant changes of pHo have also been observed during seizures and spreading depression. In the present study we have investigated regulation of V‐ATPase in hippocampal neurons following metabolic‐induced and activity‐dependent extracellular acid‐base changes. Therefore, dissociated primary hippocampal neurons and organotypic slice cultures have been used as experimental models in vitro. The results show differential regulation mechanisms of V‐ATPase depending on the cause of pathological stimulus. Metabolic acidosis caused translocation of V‐ATPase from intracellular pools towards the membrane, and increased V‐ATPase membrane expression. Acidosis‐induced V‐ATPase redistribution was mainly controlled by Rab11b, as assessed by loss‐of‐function experiments. In contrast, activity‐dependent acidosis resulted in both transcriptional and post‐translational V‐ATPase regulation in hippocampal neurons. The data suggest translolation of V‐ATPase as regulatory mechanism in neurons to cope with changes of pH and provide first evidence for a molecular mechanism underlying transporter trafficking in neurons.
An N‐terminal Signal Peptide Increases Total Expression and Enhances Surface Trafficking of Olfactory Receptors (ORs)
Authors: Shepard Blythe D , Natarajan Niranjana , Protzko Ryan J , Acres Omar W , Pluznick Jennifer L
Published: June 2021

ORs are G protein‐coupled receptors that govern the sense of smell. The majority of ORs are orphan receptors with no known ligand, largely because deorphanization typically requires OR expression on the surface of heterologous cells. Unfortunately, ORs are retained in the ER and degraded when heterologously expressed. We hypothesized that a 17‐amino acid leucine‐rich signal peptide (Lucy tag) would allow for OR surface trafficking. To test this, we added the tag to the N‐terminus of 15 ORs and assayed for surface expression by immunofluorescence (IF) of unfixed cells with an antibody against an extracellular epitope. Addition of the tag promoted surface expression of 6/15 ORs (vs. 3/15 without the tag) and increased total protein expression of all ORs as measured by immunoblotting and ELISA. When the Lucy tag was used together with a Rho tag and known OR chaperones (previously shown to aid in surface expression of a minority of ORs), all ORs reached the surface (15/15). By IF, we showed that the Lucy tag is cleaved from the immature protein and does not alter the mature protein. Consistent with this, the tag does not alter OR‐ligand binding or downstream signaling as assayed by a luciferase reporter assay for OR activation. We conclude that the Lucy tag, along with the Rho‐tag and OR chaperones, allows for the surface expression of all ORs tested, potentially allowing for future widescale deorphanization studies.

Funding: NIDDK
Organic Anion Transport in Caenorhabditis elegans
Authors: Harold Amber L , Woock Alicia E. , Bridges Andrew A. , Cecile Jennifer Perry
Published: June 2021

Organic anion transporters (OATs) aid in the elimination of negatively charged drugs and xenobiotics from the body through excretory organs such as the intestine, liver and kidney. The nonmammalian Caenorhabditis elegans (C. elegans) nematode contains one OAT isoform (CeOAT1) with sequence homology and overlapping substrate specificity to mammalian OATs. In this work, fluorescence microscopy illustrates accumulation of the organic anion fluorescein in the intestinal lumen of the wild‐type C. elegans (strain N2) and in intestinal cells in the efflux transporter knockout (strain NL152 which lacks p‐glycoprotein isoforms 1 and 3 and the multidrug resistance‐associated protein isoform 1). CeOAT1 function is investigated by quantifying fluorescein uptake in C. elegans strain NL152 with fluorescence microscopy or a fluorescence plate reader in the presence of nonfluorescent anions. Pre‐treatment with probenecid, p‐aminohippurate or folate inhibits fluorescein uptake while pretreatment with the dicarboxylate fumerate stimulates fluorescein uptake. Both agar and liquid nematode culture are amenable to these experiments and synchronized cultures provide insight into CeOAT1 function at specific life stages. These in vivo results suggest the C. elegans nematode is a model organism to examine mammalian OAT function. This research was supported by Appalachian State University.
Luminal testicular factors are essential for the luminalreaching property of basal cells in the mouse epididymis
Authors: Kim Bongki , Da Silva Nicolas , Roy Jeremy , Shum Winnie , Breton Sylvie
Published: June 2021

Basal cells (BCs) extend slender body projections that can cross the tight‐junction barrier to scan the luminal environment of the mouse proximal epididymis. We studied here the role of luminal testicular factors in the regulation of this luminal sensing property. Efferent duct ligation (EDL) was performed to block luminal flow from the testis without affecting blood flow. Keratin 5 (K5) labeling showed that the number of BCs with a luminal‐reaching projection was significantly reduced in a time dependent manner from 1 day to 5 days after EDL, compared to controls. Double labeling for caspase‐3 and K5 showed that a subset of BCs undergo apoptosis 1 day after EDL. Ki67/K5 double labeling showed an increase in the proliferation rate of the remaining BCs 2 days after EDL. In vivo treatment with the androgen receptor inhibitor, flutamide (50mg/kg/day) reduced the number of proliferating BCs in the intact tissue. We conclude that luminal testicular factors play an important role in BC luminal‐reaching ability, and are essential to maintain BC survival. Circulating androgens may play a role in the renewal of BCs following the initial insult induced by EDL. This study is funded by NIH grant DK085715.
Obesity induced infertility rescued by ovarian theca cellspecific knockout of the insulin receptor.
Authors: Wu Sheng , Wondisford Fredric , Wolfe Andrew
Published: June 2021

Many prevalent health problems are associated with obesity. Women with Type 2DM or PCOS present with elevated LH and androgen levels, anovulation and insulin resistance in metabolic tissues. The aims of these studies were to determine whether insulin signaling in the ovary played a role in the development or function of reproductive function, and to explore whether insulin exerts pathophysiologic effects at the level of the ovary in obesity. We disrupt the IR gene specifically in the theca cells of the ovaries. No changes in reproductive development or function were observed in lean KO female mice suggesting that insulin signaling in the theca cell is not essential for reproduction. To explore the effect of insulin signaling in the ovary in obesity we made mice obese (DIO) by feeding them a high fat diet. DIO WT mice were infertile and experienced increased circulating testosterone levels. By contrast, KO mice exhibited improved estrous cyclicity and fertility with comparable testosterone level as lean mice. In vitro study using a whole ovary culture model shows how HCG and insulin interact with each other to enhance androstenedione secretion. These results confirm that the high levels of insulin in obesity exert insulin action on the ovary contributing to infertility and indicate that the reproductive axis maintains insulin sensitivity even in the setting of peripheral insulin resistance.
Stress‐induced changes in gene expression associated with prostate cancer
Authors: Davalos Olinamyr , Flores Ivan , Gosselink Kristin L
Published: June 2021

Prostate cancer is common among men in the U.S., and stress is prevalent in our society, but the relationship between stress and prostate cancer is not well defined. Stress modulation of the gonadotropin‐releasing hormone (GnRH) system, and the use of GnRH analogs in prostate cancer treatment, suggests this endocrine pathway as a possible link between these two conditions. In this study, our hypothesis was that stress would suppress GnRH and increase the expression of proliferation‐associated genes in prostate tissue. Adult male rats were acutely (30min × 1d) or repeatedly (30min × 21d) restrained and prostate tissues analyzed by PCR array to determine changes in cancer‐related gene expression. Acute and repeated stress caused significant (p<0.05) downregulation of different genes associated with cellular proliferation. Angiopoeitin‐2 and cadherin‐2 were reduced by acute stress, while caspase‐2 and ‐7, IGF binding protein‐3 and ‐7, and MAPKK1 were decreased following repeated stress. Telomerase‐associated protein‐1 was significantly decreased by both stress treatments. These data demonstrate that expression of genes involved in proliferation is downregulated in prostate tissue from stressed animals. Our findings may help identify biomarkers for prostate cancer, and mechanisms through which stress may contribute to prostate cancer progression.

Support: 5U54RR022762–03S1, 8G12MD007592
Sarcolipin is a novel regulator of muscle based thermogenesis and metabolism in mammals
Authors: Bal Naresh Chandra , Maurya Santosh Kumar , Sopariwala Danesh H , Sahoo Sanjaya Kumar , Shaikh Sana A , Pant Meghna , Rowland Leslie A , Periasamy Muthu
Published: June 2021

Sarcolipin (SLN) is a novel regulator of Sarco/Endoplasmic Reticulum Ca2+‐ATPase (SERCA) pump and it is expressed exclusively in striated muscle. Recent in‐vitro studies suggested that SLN can increase heat production by uncoupling SERCA‐mediated ATP hydrolysis from Ca2+ transport but its role in muscle physiology has not been established. To demonstrate that SLN is the basis for muscle thermogenesis in vivo, we challenged SLN−/− mice to acute cold (4°C). Here we show that SLN−/− mice without interscapular Brown Adipose Tissue (iBAT) failed to maintain core body temperature (Tc, 37°C) and died when exposed to acute cold (4°C), whereas wildtype (WT) mice without iBAT were able to maintain Tc. Overexpression of SLN in the null background fully restored muscle‐based thermogenesis, suggesting that SLN is the basis for SERCA‐mediated heat production. We found that curare mediated blockade of shivering in WT mice without iBAT did not significantly compromise thermogenesis indicating existence of non‐shivering thermogenesis (NST) in muscle. We show that Ryanodine receptor (RyR1)‐mediated Ca2+ leak is an important mechanism for SERCA‐activated heat generation. We also provide experimental data to suggest that SLN can interact with SERCA in the presence of Ca2+ and is thereby capable of promoting uncoupling of the SERCA pump and increasing heat production. We further demonstrate that loss of SLN predisposes mice to diet induced obesity, which suggests that SLN mediated NST is recruited during metabolic overload. These data collectively suggest that SLN is an important mediator of muscle thermogenesis and this mechanism could be recruited in energy metabolism.
Histone deacetylase‐dependent control of lung alveolar fluid clearance
Authors: Wujak Lukasz Andrzej , Becker Simone , Arnoldt Verena , Wygrecka Malgorzata , Seeger Werner , Morty Rory Edward
Published: June 2021

TGF‐β mediates acute lung injury (ALI) by impacting alveolar ion transport and thereby promoting pulmonary edema. The Na,K‐ATPase regulates ion transport and fluid balance and Na,K‐ATPase function is perturbed in ALI. It was hypothesized that TGF‐β impacts Na,K‐ATPase activity by altering subunit stoichiometry. Na,K‐ATPase activity and surface abundance were reduced in primary alveolar type II and A549 cells treated with TGF‐β, assessed by 86Rb+ uptake and cell surface biotinylation. The ATP1B1 subunit regulates surface stability, and hence alveolar fluid clearance (AFC) activity of the Na,K‐ATPase. PCR analysis revealed repression of ATP1B1 gene expression in lungs from ALI patients, in mice with bleomycin‐induced ALI model and in TGF‐β‐treated A549 cells. A Dual‐luciferase reporter assay revealed decreased ATP1B1 promoter activity in response to TGF‐ β. Enzyme inhibition and siRNA approaches demonstrated that ATP1B1 gene repression was mediated by epigenetic mechanisms and relied on HDAC2. TGF‐β induced phosphorylation of HDAC2 at S394 while chromatin immunoprecipitation confirmed binding of the HDAC2 to the ATP1B1 promoter. HDAC inhibition abrogated repression of the Atp1b1 gene in bleomycin‐induced ALI and drove edema resolution. In sum, HDAC2 mediated repression of the ATP1B1 gene, impairing Na,K‐ATPase function and AFC in ALI.

German Research Foundation.
Analysis of Cardiac Mitochondrial Proteome Dynamics Shows Decreased Rate of Protein Synthesis in Heart Failure
Authors: Shekar Kadambari Chandra , Li Ling , Dabkowski Erinne , Xu Wenhong , Ribeiro Rogerio Faustino , Willard Belinda , Stanley William C , Kasumov Takhar
Published: June 2021

We developed a novel method to assess the synthesis rate of individual mitochondrial proteins using deuterium labeled water (D2O) as a stable isotopic tracer and advance mass spectrometric analysis. Here we assess the impact of heart failure (HF) on mitochondrial protein synthesis. HF was induced in rats by pressure overload with transverse aortic constriction for 22 weeks. Individual protein synthesis rates were assessed by giving 5% D2O in drinking water for up to 80 days, and measuring deuterium incorporation into mitochondrial proteins. There was clear HF, with 44% increase in left ventricular (LV) mass, and 40% decrease in LV fractional shortening compared to shams. Subsarcolemmal mitochondria were isolated. State 3 respiration decreased by 16% with HF using palmitoyl carnitine as the substrate, but was unaffected with non‐lipid substrates. Mitochondrial protein synthesis rates were measured by LTQ Orbitrap MS. Initial analysis showed that the fatty acid oxidation (FAO) enzyme trifunctional protein alpha subunit had a half‐life (t1/2) of 29 days in shams, which increased to 41 days with HF. The t1/2 of the electron transport chain (ETC) protein complex 3 core 2 subunit increased dramatically from 76 days in shams to 166 days in TAC rats. In conclusion, HF induced by cardiac hypertrophy slows the synthesis rate of key mitochondrial proteins, with a greater effect on ETC proteins than on FAO proteins.