bluuebunny/crossref_metadata_2025 huggingface dataset:
G protein‐coupled receptor (GPCR) regulation of cardiac fibrosis
Authors: Snead Aaron Nathan , He Steven , Insel Paul A.
Published: June 2021

One form of heart disease, cardiac fibrosis, is characterized by transformation of cardiac fibroblasts (CFs) to pro‐fibrogenic myofibroblasts, which produce excessive amounts of collagen‐rich extracellular matrix, thereby reducing cardiac function and eventually resulting in heart failure. Certain GPCRs, e.g. angiotensin receptors, can promote cardiac fibrosis but few others have been studied in this context. To identify novel therapeutic targets for cardiac fibrosis we used an unbiased approach (GPCR RT‐PCR array) to define the most abundantly expressed GPCRs that may function to regulate the fibroblast/myofibroblast transformation in rat CFs (rCFs). We identified protease‐activated receptor 1 (PAR1) as the most abundant GPCR and found that activation of PAR1 elevates the expression of numerous profibrotic markers, increases collagen synthesis, and alters cell morphology of rCFs. Additionally, since analogs of the second messenger cAMP can inhibit the phenotype induced by pro‐fibrotic agents (e.g. TGF‐β1 and angiotensin II), we have assessed rCF responses to the increase in cAMP produced by two abundantly expressed Gs‐linked GPCRs, adenosine A2A and A2B receptors. These data, which define the level of expression of GPCRs in rCFs and mechanisms for their actions, provide a proof‐of‐principle that such an approach can identify novel disease‐relevant GPCR targets. Supported by NIH.
MMP‐9 Generated Collagen I C‐propeptides Alter Cardiac Fibroblast Function
Authors: de Castro Bras Lisandra E , Dai Qiuxia , Zamilpa Rogelio , Fields Gregg B , Weintraub Susan T , Lindsey Merry L
Published: June 2021

Heart failure is the result of adverse remodeling of the collagenous scar that replaces the damaged myocardium after myocardial infarction (MI). Post‐MI, specific matrix metalloproteinases (MMPs) increase and mediate left ventricle (LV) remodeling. Of the MMPs family members, MMP‐9 levels have been correlated with cardiovascular mortality in humans. Animal models show that MMP‐9 levels increase early post‐MI, and MMP‐9 deletion in mice attenuates remodeling. We found that C‐terminal propeptide (C‐propeptide) levels of á2(I) collagen were reduced in MMP‐9 null mice. Also, we have recently found a novel MMP‐9 recognition site that generates a C‐propeptide of 30 kDa.

We used increasing doses of collagen fragments from the C‐terminus domain to stimulate cardiac fibroblasts and determine the mechanisms whereby C‐ propeptides regulate collagen function. We identified one fragment that strongly enhanced collagen I synthesis, suggesting a positive feedback loop between collagen processing and collagen synthesis. These results indicate that in addition to mediating ECM degradation, MMP‐9 may also regulate collagen I synthesis post‐MI.
Matrix Metalloproteinase‐28 Deletion Attenuates Short‐term Left Ventricular Dysfunction but Exacerbates Cardiac Rupture Post‐Myocardial Infarction in Mice
Authors: Ma Yonggang , Zhang Jianhua , Ramirez Trevi A. , Manicone Anne M. , Lindsey Merry L.
Published: June 2021

Matrix metalloproteinase (MMP)‐28 is expressed in normal adult hearts and is also expressed in macrophages suggesting a role in cardiac wound healing. The significance of MMP‐28 in myocardial infarction (MI), however, has not been explored. Adult C57BL/6 wild type (WT) and MMP‐28−/− mice were subjected to left coronary artery ligation and sacrificed at multiple time points up to day 7 post‐MI. Immunofluorescence showed that MMP‐28 was expressed by cardiomyocytes under normal conditions and by macrophages post‐MI. Ejection fraction at day 1 post‐MI was markedly depressed in WT (17±2%), and this impaired function was attenuated in the MMP‐28−/− mice (23±1%; p<0.05, n=9/group). The 7 day survival rate after MI for WT was 59% (13/22) and for MMP‐28−/− was 30% (10/33; p<0.05). Autopsy data revealed that 33% of WT mice died from left ventricular (LV) rupture; however, the rupture rate in MMP‐28−/− mice was 87% (p<0.05). At day 5 post‐MI, the mRNA levels of 14 out of 84 inflammatory genes were statistically increased in the infarct regions of the MMP‐28−/− mice, compared to WT (all p<0.05). Included genes were Casp1, Ccl2, Ccl7, Ccl12, Ccr5, Ccr7, Ccr9, Cxcr3, Cxcr5, Il2rg, Il10ra, Ltb, Tgfb1 and Tnfrsf1b. In conclusion, MMP‐28 deletion blunts short‐term LV dysfunction, but aggravates cardiac rupture by exacerbating the pro‐inflammatory response.
Increased MMP8 and 12 activation correlates with elevated endostatin and angiostatin and impaired coronary collateral growth in the metabolic syndrome
Authors: Dodd Tracy Y. , Wiggins Luke , Musiyenko Alla , Smith Erika , Rocic Petra
Published: June 2021

We have previously shown that repetitive ischemia (RI) stimulated coronary collateral growth (CCG) in healthy (SD) but not metabolic syndrome (JCR) rats, and that matrix metalloproteinases (MMPs) play a critical role in the regulation of CCG by extracellular matrix (ECM) degradation. We hypothesized that MMPs may also negatively regulate CCG via the release of anti‐angiogenic peptides, endostatin and angiostatin, generated during ECM degradation. To generate RI, an occluder was implanted over the LAD, and rats underwent the RI protocol (8 40 sec occlusions; every 20 min, every 8 hrs for 0, 3, 6, or 9 days). CCG was measured in the ischemic, collateral‐dependent and the normal zones by microspheres, MMP8 and 12 expression and activation by Western blot, and endostatin and angiostatin levels by ELISA on days 0, 3, 6 and 9 of RI. Endostatin and angiostatin were highly increased in JCR but not in the SD rats on days 6 and 9 of RI. Increased endostatin and angiostatin levels correlated with a significant increase in MMP8 and 12 activation in JCR on days 6 and 9 of RI, which were not observed in the SD rats. We conclude that compromised CCG in the metabolic syndrome may be partially due to increased MMP8 and 12 activation and consequent increased generation of anti‐angiogenic peptides, which may preclude collateral vessel expansion. Support: RO1 HL093052
Transfat‐mediated apoptosis is regulated by autophagy in primary cardiac myofibroblasts
Authors: Rattan Sunil , Ghavami Saeid , Cunnington Ryan H , Davies Jared L , Bathe Krista L , Yeganeh Behzad , Arora Rakesh , Los Marek J , Freed Darren H , Halayko Andrew J , Klonisch Thomas , Pierce Grant N , Dixon Ian MC
Published: June 2021

Dietary trans fatty acids (TFA) are linked to cardiovascular disease which has led to recommendations to remove TFAs from diet. We investigated the effects of two common fatty acids ‐ vaccenic acid (VA ‐ found in dairy products), and elaidic acid (EA ‐ found in hydrogenated vegetable oils) on cell death in primary rat ventricular myofibroblasts (rVF). VA‐ and EA‐treated cells exhibited apoptotic cell death in rVF, as reflected in MTT and FACS assays. A significant reduction in mitochondrial membrane potential and an increase in the Bax/Bcl2 ratio and Bax translocation to the mitochondria was found suggesting apoptosis in the presence of TFA. Elevated cleaved caspase‐9 and caspase‐3 (without caspase‐8), shows that EA and VA treatments induce caspase‐dependent apoptosis. Transmission electron microscopy reveals autophagosome formation and lysosome activation in VA and EA treated rVF. Induction of autophagy markers including LC3‐B lipidation, increased Atg5‐12 formation and increased beclin‐1 were observed confirming autophagy activation by EA and VA. Inhibition of ATG3 and ATG5 significantly inhibited cytotoxic effects by preventing caspase‐3 and caspase‐7 activation in EA and VA treated rVF. In conclusion we show that TFAs induce apoptosis and autophagy in rVF and that TFA‐induced autophagy is required for this proapoptotic effect. Supported by CIHR and DFC.
LECTINIC BINDING OF CANDIDA GLABRATA TO OLIGOSACCHARIDES ON THE CORONARY LUMINAL MEMBRANE ALTERS FLOW‐INDUCED CARDIAC RESPONSES
Authors: Torres-Tirado David , Ramirez-Zavaleta Candy , De Las Peñas Alejandro , Castaño Irene , Rubio Rafael
Published: June 2021

Candida Glabrata (CG) is a yeast that may invade humans blood stream causing dead. CG surface is lectinic and binds with high affinity to oligosaccharides (O) present in cell membranes, as example, coronary endothelial luminal membrane (CELM).

Conversely, coronary flow (CF) stimulates auricular‐ventricular transmission (A‐Vt). CF acts on CELM lectins (L) and O, causing a reversible L‐O complexing necessary for CF stimulation of A‐Vt. We hypothesized that CG upon binding to CELM's O prevents CF‐induced L‐O complexing; as a result inhibiting CF‐stimulation of A‐Vt.

In Langendorff perfused isolated guinea pig hearts A‐Vt was determined at different CF. As CF (ml/min) increase A‐Vt (msec) decrease. (control curve). Thereafter, to study the effects of CG bound to the CELM, CG (CGM35 sir3Δ, 3.7×103 cells/ml), was intracoronary given for 5 min and wash at length for 5 min and an experimental CF‐ A‐Vt curve determined. The experimental curve was on top of the control curve i. e. CG upon binding to CELM increases the A‐Vt value at any CF. To test the effect was due to CG bound to O, CG was displaced by perfusing sugars components of O; mannose or Galactose. Perfusion of any of these sugars after CG they reverted its inhibitory effects on CF‐induced A‐Vt stimulation. Furthermore, simultaneous perfusion of CG and any of these sugars prevented the CG‐inhibitory effects, likely competing with CELM's O for CG binding. Thus, CG upon binding to CELM's O competes with L preventing CF‐induced L‐O complexing; as a result inhibits CF‐stimulation of A‐Vt.
Allergen‐induced airway constriction in guinea pig lung slices is attenuated by arginase inhibition via increased nitric oxide production
Authors: Sopi Ramadan B , Simoons Mirjam , Sanderson Michael J , Meurs Herman , Maarsingh Harm
Published: June 2021

In a guinea pig model of allergic asthma, arginase inhibition protects against allergen‐induced airway obstruction and greatly (>30‐fold) reduces airway sensitivity towards the inhaled allergen. Since arginase regulates nitric oxide (NO) synthesis via competition with NO synthase (NOS) for the common substrate L‐arginine, we hypothesized that the anti‐allergic effect of arginase inhibition involves increased NO synthesis. Ovalbumin (OVA; 0.0001–100 μg/ml)‐induced airway constriction was measured in lung slices obtained from actively‐sensitized guinea pigs using video‐assisted microscopy, in the absence or presence of inhibitors of arginase (ABH, 1μM or nor‐NOHA, 5 μM) and/or NOS (L‐NAME, 100 μM). OVA induced a dose‐dependent airway constriction with a mean maximal effect (Emax) of 98% and a mean EC50 of 0.039 μg/ml. ABH significantly reduced the Emax of OVA to 50%, whereas the sensitivity towards OVA was reduced by >100‐fold, to 5 μg/ml. nor‐NOHA mimicked the effects of ABH, whereas L‐NAME slightly increased contractile responses towards OVA (Emax: 100%; EC50: 0.007 μg/ml). L‐NAME partially reversed the protective effect of ABH against OVA (Emax: 74%; EC50: 0.125 μg/ml). In conclusion, arginase inhibition reduces allergen‐induced contractile responses of intrapulmonary airways by increasing NO synthesis.

Supported by Erasmus Mundus partnership Join EU‐SEE and NIH grant HL‐103405.
Effects of latrunculin‐B in smooth muscle contraction, Ca2+ oscillations and Ca2+ sensitization in small intrapulmonary airways
Authors: Trice Jacquelyn , Perez-Zoghbi Jose F
Published: June 2021

The role of actin filament dynamics in the regulation of airway smooth muscle contraction is not fully understood. We have investigated the effects of impairing actin filament polymerization with latrunculin‐B (Lat‐B) –a G‐actin binding toxin, on agonist‐induced Ca2+ signaling, Ca2+ sensitivity and contraction in small intrapulmonary airways in mouse lung slices. Incubation of lung slices with Lat‐B (0.1 μM) for 60 min caused a reduction of airway contraction induced by 0.1 μM serotonin (5‐HT) and 0.1 μM acetylcholine (ACh) by 54 ± 8% and 42 ± 6%, respectively. In addition, Lat‐B induced relaxation of airways pre‐contracted with agonists. Maximal airway relaxation was 55 ± 5% and 53 ± 8% in airways pre‐contracted with 5‐HT and ACh, respectively. Furthermore, Lat‐B affected the Ca2+ signaling induced by ACh and 5‐HT by reducing the frequency of agonists‐induced Ca2+ oscillations. Finally, in Ca2+ permeabilized lung slices, Lat‐B induced relaxation of airways pre‐contracted with either 5‐HT or ACh indicating Ca2+ desensitization. Together these results suggest that actin filament dynamics are important for the regulation of airway smooth muscle contraction and signaling.
Respiratory Syncytial Virus Infections Augment Airway Reactivity in Mice Exposed to Chlorine (Cl2)
Authors: Song Weifeng , Yu Zhihong , Ambalavanan Namasivayam , Garantziotis Stavros , Matalon Sadis
Published: June 2021

Chlorine (Cl2) gas inhalation causes respiratory system injury including epithelial cell sloughing, edema and airway hyperreactivity, leading to reactive airway dysfunction syndrome (RADS), a prelude to asthma. However, little is known about how pre‐existing infections alter the responses of animals to a subsequent Cl2 exposure. Herein Balb/c mice were infected with respiratory syncytial virus (RSV), a common respiratory infection in children, via intranasal instillation of 100 microliters of RSV A2 (106 PFU/ml) or vehicle. Four days later they were exposed to 187 ppm Cl2 for 30 minutes in environmental chambers and returned to room air. Measurements of airway resistance and elastance, prior to and following challenge with aerosolized methacholine (up to 50 mg/ml) were conducted in anesthetized mice with a flexiVent, 24 h post exposure. RSV infection greatly increased: (1) airway resistance and elastance after methacholine challenge in Cl2 exposed mice as compared to vehicle infected mice; (2) levels of IL‐10, interferon‐γ and RANTES and (3) levels of low molecular weight hyaluronan and inter‐α‐trypsin inhibitor (IaI; a factor necessary for the binding of hyaluronan to its receptor) in the BAL. Hyaluronan has been shown to contribute to airway hyperresponsiveness in mice exposed to ozone. We concluded that pre‐existing viral infections increase the susceptibility of mice to Cl2 induced airway injury via the upregulation of a variety of inflammatory cytokines and hyaluronan.
Perinatal Inflammation and Decreases in miR29b‐1 Expression are Associated with Structural and Functional Pulmonary Deficits in Adult Mice
Authors: Velten Markus , Britt Rodney D , Heyob Kathryn M , Welty Stephen E , Tipple Trent E , Rogers Lynette K
Published: June 2021

Background

Maternal inflammation contributes to premature delivery. Premature infants routinely receive therapeutic oxygen and exhibit persistently diminished respiratory function into adulthood. The long term pulmonary consequences of perinatal inflammation are unclear.

Objective

We tested the hypothesis that a hostile perinatal environment induces fibrotic pathways resulting in pulmonary fibrosis.

Methods

Pregnant C3H/HeN mice were LPS or saline injected on embryonic day (E) 16. Offspring were placed in room air (RA) or 85% O2 for 14 d then returned to RA. PFTs, microCTs, molecular, and histological analyses were performed between E18 and 8 weeks of age.

Main Results

Alveolarization was most compromised in LPS/O2 exposed mice. Collagen staining and protein levels were increased and static‐compliance was decreased only in LPS/O2 exposed mice. 3‐D MicroCT reconstruction revealed increased tissue densies only in LPS/O2 mice. Development of fibrosis was associated with decreased miR29b‐1 expression and evidence of TGF‐β/Smad signaling during embryonic and early fetal lung development.

Conclusions

Systemic maternal LPS administration induces a pro‐fibrotic response in fetal lung tissue that in combination with neonatal hyperoxia exposure depresses miR29b‐1 and activates of pro‐fibrotic pathways, resulting in impaired alveolarization, fibrosis, and compromised function in adulthood.
The hyperoxia‐mediated decrease in tenascin‐C in fetal lung fibroblasts inhibits cell migration
Authors: DeCoux Ashley , Chaplin Jennifer , Wilson Glenn , Benjamin John , Gebb Sarah
Published: June 2021

Hyperoxic exposure is associated with the arrested alveolarization characteristic of bronchopulmonary dysplasia (BPD). The myofibroblast is required for normal alveolarization and synthesizes important extracellular matrix (ECM) proteins. In a reciprocal manner, this ECM modulates fibroblast function. To investigate the link between ECM and lung myofibroblast migration, we tested the hypothesis that hyperoxia inhibits fetal lung fibroblast TN‐C expression, leading to a reduction in cell migration. Primary fetal lung fibroblasts were maintained at fetal O2 tension (3% O2) and transitioned to hyperoxia (21%, 40%, and 65% O2) for 48 hours. Hyperoxia exposure reduced TN‐C protein expression in a dose‐dependent manner, with a >10‐fold decrease in TN‐C protein in fibroblasts grown in 65% O2. In contrast, the ECM protein fibronectin (FN) was unchanged by hyperoxia exposure. Functionally, the hyperoxia‐mediated decrease in TN‐C was accompanied by a 40% reduction in cell migration. We speculate that the anti‐adhesive properties of TN‐C promote fibroblast migration in the developing lung, and hyperoxia‐induced loss of TN‐C inhibits this migration by promoting cell adhesion.
Cell‐specific expression of alveolar TNFR1
Authors: Gusarova Galina A , Islam Mohammad Naimul , Das Shonit , Bhattacharya Jahar
Published: June 2021

Ligation of the cytokine, tumor necrosis factor alpha (TNFα) to TNFα receptor 1 (TNFR1) causes lung inflammation. Therefore, the organization and extent of TNFα‐induced lung inflammation is likely to depend on the distribution and density of TNFR1 in the alveolar epithelium (AE). We addressed this issue by real‐time confocal microscopy of intact alveoli in the isolated, blood‐perfused mouse lung (n=3). To visualize the AE, we microinjected alveoli with the cytosolic marker, calcein green, and with LysoTracker Red (LTR) which localizes to lamellar bodies in alveolar type 2 (AT2) cells. To determine TNFR1 expression, we injected an Alexa‐633‐conjugated anti‐TNFR1 mAb that recognizes an extracellular domain of mouse TNFR1. TNFR1 fluorescence was accepted at ≥50 grey levels. In the imaged region, each alveolus contained 1‐2 AT2 cells, consistent with the understanding that the majority of the alveolar surface area is provided by alveolar type 1 (AT1) cells. TNFR1 expression was spatially heterogeneous, since the receptor fluorescence co‐localized with AT1, but not AT2 cells (P<0.05). Our findings suggest that the primary alveolar inflammatory response to TNFα is driven by TNFR1‐induced signaling in the AT1 cell (HL57556, HL64896).
Cigarette smoke increases susceptibility to lung edema: Implication of RhoA and FAK‐mediated disassembly of endothelial cytoskeleton and cell contacts
Authors: Lu Qing , Sakhatskyy Pavlo , Newton Julie , Rounds Sharon
Published: June 2021

Cigarette smoke (CS) is a major risk factor for chronic lung and heart diseases. Whether CS is a risk factor for acute lung injury (ALI) remains controversial. Using mice exposed to acute CS and cultured lung endothelial cells exposed to cigarette smoke extract (CSE), we have shown that CS causes endothelial barrier dysfunction and exacerbates lipopolysaccharide (LPS)‐induced increase in vascular permeability in vitro and in vivo. CSE‐induced barrier dysfunction is mediated via oxidative stressinduced inhibition of RhoA and focal adhesion kinase (FAK). The aim of this study is to further address the effect and mechanism of CS on susceptibility to ALI. We found that similar to acute exposure, chronic CS exposure, which caused mild emphysema, increased susceptibility to LPS‐induced lung edema. However, chronic CS exposure alone did not increase lung vascular permeability. We also found that CSE disrupted focal adhesion complexes (FAC), F‐actin fibers and adherens junctions (AJ) via inhibition of RhoA and FAK. In addition, CSE caused disassembly of microtubule (MT) and MT stabilizer, taxol, blunted CSE‐induced barrier dysfunction. Our data suggest that CS causes endothelial barrier dysfunction via disassembly of cytoskeleton and cell contacts. Increased susceptibility to lung edema may be yet another health consequence of CS. Supported Finding: VA Merit Review (Rounds), HL64936 (Rounds), ATS/PHA grant (Lu)
Fibronectin stabilization and Fibronectin‐Integrin signaling via MAPK are required in L‐Glutamine‐mediated protection against gut injury
Authors: Niederlechner Stefanie , Klawitter Jelena , Baird Christine , Kallweit Alyssa , Christians Uwe , Wischmeyer Paul
Published: June 2021

We assessed if Fibronectin (FN) expression and FN‐Integrin pathways, which play a protective role after gut injury, are involved in L‐Glutamine's (GLN) protective mechanism in the intestine after heat stress (HS). IEC‐6 cells were treated with 0mM or 10mM GLN with or without FN siRNA (20nM), integrin inhibitor GRGDSP (50μM), control peptide GRGESP (50μM), or ERK1/2 inhibitor PD98059 (50μM). Cell survival was measured via MTS assay 24h post‐HS (44ºCx50min). Total ERK1/2 and HSF‐1, [T(P)202/Y(P)204]ERK1/2, [S(P)303]HSF‐1, HSP32, HSP70, FN, cleaved caspase‐3 and PARP levels were determined via Western blot, and GLN‐transport via LC‐MS/MS after non‐lethal HS (43ºCx45min). GLN prevented the decrease in FN levels after HS and GLN's protection decreased after FN siRNA treatment (p<0.05; n=3). Thus, GRGDSP did not affect GLN transport into the cells (p<0.05; n=3), GRGDSP and PD98059 attenuated GLN‐mediated protection, shown by MTS, cleaved caspase‐3 and PARP levels (p<0.05; n=5). GRGDSP abolished GLN‐mediated Erk1/2 activation after HS (p<0.05; n=4). GLN activated HSF‐1 by attenuating its’ phosphorylation at serine 303 (p<0.05; n=3), but HS, HS+GRGDSP+GLN and HS+PD98059+GLN repressed HSF‐1 by its’ phosphorylation (p<0.05; n=3). GLN‐mediated increases in HSP70 and HSP32 were attenuated by GRGDSP after HS (p<0.05; n=4). FN stabilization and FN‐Integrin signaling via MAPK by GLN seem to be vital after gut injury.
Expression of gene isoforms encoding phospholipase C X‐domain containing proteins in fish and humans
Authors: Gellatly Steven , Kalujnaia Svetlana , Cramb Gordon
Published: June 2021

Comparative analysis of microarrays data from intestine of sexually immature “yellow” eels and sexually mature “silver” eels revealed the differential expression of a novel member of the phospholipase C (PLC) family, known as PLCX‐domain containing protein (PLCXD), which has unknown function. We investigated the expression of the PLCXD gene in eel, zebrafish and human. The expression of three human PLCXD isoforms known as ‐XD1, ‐XD2 and –XD3 have been characterised in tissues and cultured cell lines. The proteins exhibit differential cellular location (when expressed in HeLa cells) suggesting different functions. PLCXD1 is expressed in perinuclear vesicles, ‐XD2 in the nucleus, and –XD3 in the cytoplasm. In zebrafish we identified five PLCXD isoforms. The eel PLCXD protein shares maximum of similarity (51%) with zfPLCXD4 and only around 40% with all hPLCXDs. In contrast, zfPLCXD1, 2, and 3 orthologues share 65%, 68%, and 93% aa. homology with the human equivalents. Zebrafish and human PLCXD gene isoforms show similar tissue expression. For example, all are expressed in the brain and PLCXD3 is mostly expressed in heart. There is also difference in gene expression between species, eg. the highest level of eel PLCXD expression is in intestine, but the equivalent homologue in zebrafish is mainly in the head kidney. Our studies indicate different functional roles of PLCXD proteins during evolution.
Effect of Ketanserin and Methiothepin Implicate a 5‐HT‐like Receptors in Serotonin‐stimulated Alkali and Acid Secretion in Isolated, Perfused Midgut Preparations of Larval Yellow‐fever Mosquito Aedes aegypti
Authors: Cobbina Alphonse Karl , Moffett David F.
Published: June 2021

Previous work from our laboratory has shown that serotonin (5‐HT) is a major control factor for alkalinization of the anterior midgut (AMG) and acidification of the posterior midgut (PMG) of the larval mosquito, Aedes aegypti. Heretofore it has been impossible to associate the effect of serotonin on acid‐base transport with particular 5‐HT receptor(s) in the larval mosquito gut. Ketanserin (10μM), a specific antagonist for 5‐HT2‐like receptors, was effective against 5‐HT stimulation of alkali secretion by the AMG, but not the acid secretion by the PMG, whereas Methiothepin (10μM) which is active against 5‐HT1, 5‐HT6 and 5‐HT7 but not 5‐HT2‐like receptors, was ineffective against alkali secretion by the AMG but effective against acid secretion in the PMG. Since 5‐HT2 receptors are expected to act through an IP3/DAG pathway to secrete cytoplasmic Ca2+. This result is in accord with our recent demonstration that a Ca2+ 2nd message is required for initiation of 5‐HT stimulated alkali and acid secretion.
TRPV4 in Porcine Lens Epithelium Regulates Hyposmotic Stress‐Induced ATP Release and Na, K‐ATPase Activity
Authors: Shahidullah Mohammad , Mandal Amritlal , Delamere Nicholas A
Published: June 2021

In several tissues TRPV4 channels are involved in the response to hyposmotic challenge. Our objective was to detect TRPV4 protein in porcine lens epithelium and its role in hyposmotic stress‐induced ATP release.

The Western blot analysis showed presence of TRPV4 in the lens epithelial homogenate. The TRPV4 antagonist RN 1734 (10μM) completely prevented ATP release by lenses exposed to hyposmotic (200 mOsm) solution. Lenses exposed to a TRPV4 agonist, GSK1016790A (GSK), or to hyposmotic solution displayed ATP release and an increased ability of propidium iodide (PI) (MW 668) to enter the epithelium. The increases in PI uptake and ATP release both were abolished by a mixture of agents that block connexin and pannexin hemichannels, 18á‐glycyrrhetinic acid (AGA, 100μM) and probenecid (1mM). An increase in Na, K‐ATPase activity was detected in the epithelium of lenses exposed to GSK in isosmotic solution and the response was prevented by AGA + probenecid. TRPV4 antagonists prevented the activation of Src family kinase and increase of Na, K‐ATPase activity that occurred in the epithelium of lenses exposed to hyposmotic solution.

The findings are consistent with hyposmotic shock‐induced TRPV4 channel activation which triggers hemichannel‐mediated ATP release. The results point to a pivotal role for TRPV4 activation as an initial event in a multi‐step response of the lens to hyposmotic stress.
Ubiquitination of Vesicular Acetylcholine Transporter
Authors: Li Yifan , Freeling Jessica , Sample Alex
Published: June 2021

Vesicular acetylcholine transporter (VAChT) is responsible for transporting synthesized acetylcholine (Ach) into synaptic vesicles and thus affects Ach release at cholinergic synapses. Trafficking and distribution of VAChT in cells involves complex processes which are not fully understood. Utilizing Tandem Ubiquitin Binding Entities (TUBE) assay, we pulled down the ~70 kDa band of VAChT in samples from the SN56 cholinergic cell line, as well as from mouse brain tissues, indicating this is the ubiquitinated specie of VAChT. In SN56 cells, this ubiquitinated VAChT was increased by the treatment with a non‐selective ubiquitin isopeptidase inhibitor, PR‐619, but decreased by the treatment with an ubiquitin activating enzyme (E1) inhibitor, PYR‐41. Furthermore, manipulations of VAChT ubiquitination impacted cytosolic and membrane distribution of this transporter. Collectively, these data suggest that ubiquitination may play a role in regulating distribution and thus function of VAChT in cholinergic neurons.

This work was supported by AHA grant No.0835256N, NIH INBRE grant No. P20 RR016479, NIH grant No. 1R03AG033291, and the BBS bridge grant.
Mice disrupted for the ammonia channel RhCG compensate a physiological acid load of high protein diet
Authors: Bounoure Lisa , Bourgeois Soline , Druart Sébastien , Kuhn Gisela , Müller Ralph , Devuyst Olivier , Wagner Carsten
Published: June 2021

Ammonium (NH4+) excretion in the urine is a critical mechanism by which the kidneys clear the excess of acid from the blood. A breakdown in this process leads to metabolic acidosis, as it was observed in HCl acid‐loaded mice harboring a deletion of the ammonia channel RhCG gene. Dietary intakes enriched in protein are known to induce an acid load in both human and rodents. We used here a high protein diet (HP) to investigate the response of Rhcg +/+, +/− and −/− mice exposed to a mild physiological acid load. Metabolic parameters of these animals treated for 9 days with HP diet revealed that after 4 days, Rhcg −/− excreted 30% less urinary NH4+ than +/+, but at day 9, Rhcg −/− mice interestingly adapted fully to the diet, excreting as much NH4+ as Rhcg +/+. The mRNA and protein expression of proteins involved in renal NH4+ metabolism, SNAT3, PDG, and NKCC2 were enhanced in Rhcg +/− or −/− and both groups concentrated more NH4+ in the inner medulla than Rhcg +/+, suggesting that these mice produce and reabsorb more NH4+ to properly acidify urine after HP acid load. Finally, Rhcg −/− presented signs of bone degradation with hypercalcemia and hypercalciuria after 4 days of HP diet together with an increased release of deoxypiridinoline (a marker of bone resorption). Thus, Rhcg −/− and +/− mice handle a physiological HP acid load using specific adaptation mechanisms, however they cannot avoid bone degradation under these conditions.

Supported by the Swiss National Science Foundation
Secondary hyperparathyroidism and impaired renal phosphate excretion in mice lacking adenylyl cyclase 6
Authors: Rieg Timo , Tang Tong , Sharik Meaghen , Hammond H Kirk , Vallon Volker
Published: June 2021

Parathyroid hormone (PTH) activates an unidentified isoform of adenylyl cyclase (AC) thereby retrieving Na+/phosphate cotransporters from the proximal tubule membrane and increasing phosphate (Pi) excretion. Fibroblast growth factor (FGF) 23 is a novel AC‐independent phosphaturic hormone. To test for a role of AC6 we studied wild‐type (WT, n=10) and AC6 knockout (AC6−/−, n=9) mice under different Pi intake (low: <0.02% Pi; control: 0.6% Pi; both 0.8% Ca2+). Plasma Pi was similar on control diet (both 1.6±0.1 mM) but AC6−/− had lower urinary Pi/creatinine (Crea) (7±1 vs 17±1 mM/mM, P<0.05) despite of higher plasma PTH and FGF23 vs WT (1057±223 vs 140±23 and 923±124 vs. 629±119 pg/ml, P<0.05). Low Pi intake (7 days) suppressed PTH (11±5 and 7±7 pg/ml); despite of higher FGF23 (629±119 vs 302±78 pg/ml, P<0.05) AC6−/− had lower urinary Pi/Crea (0.09±0.01 vs 0.13±0.01 mM/mM, P<0.05) and maintained higher plasma Pi vs WT (1.3±0.1 vs 0.9±0.1 mM, P<0.05). Acute oral phosphate loading (0.5 M NaH2PO4, 1% of bw) under low Pi diet induced higher plasma Pi in AC6−/− vs WT (Δ change 30 min: 1.3±0.2 vs 0.8±0.1 mM; 60 min: 2.1±0.3 vs 1.4±0.2 mM, P<0.05) but urinary Pi/Crea tended to be lower in AC6−/− (60 min: 35±14 vs 63±16 mM/mM). Dissociation of PTH and urinary Pi excretion in AC6−/− is consistent with AC6 mediating PTH‐induced inhibition of renal Pi reabsorption, while upregulation of FGF23 may compensate.
Tracking of single microvilli to study regulation of the intestinal phosphate transporters
Authors: Lanzano Luca , Caldas Yupanqui , Giral Hector , Digman Michelle , Levi Moshe , Gratton Enrico
Published: June 2021

Intestinal phosphate (Pi) uptake is a key mechanism in systemic Pi homeostasis. Modulation of intestinal Pi transport has been recently recognized as an important target in prevention of hyperphosphatemia and the associated cardiovascular complications in Chronic Kidney Disease. Intestinal Pi absorption is mainly mediated by the sodium‐phosphate co‐transporter NaPi2b expressed in the intestinal apical microvilli. Recent evidences show that agonists of the LXR nuclear hormone receptor inhibit intestinal NaPi transport activity, however, the detailed mechanisms of how LXR decreases intestinal NaPi transport activity, including the role of changes in membrane cholesterol or PDZ interacting proteins, remain to be determined.

We study these processes at the molecular level applying the Modulation Tracking nanoimaging method to microvilli of live cells expressing fluorescently tagged NaPi2b. Tracking single microvilli we measure their motion, image NaPi2b distribution on the membrane and perform Fluorescence Correlation Spectroscopy techniques (RICS, N&B) in steady state, even if the microvilli are moving. Motility of the microvilli, protein diffusion and clustering, are monitored under stimulation to get insights into the process of intestinal transporters regulation.

Work supported in part by NIH R01 DK066029‐01A2 (ML, EG and LL), NIH‐P41‐RR03155 (EG and LL) and P50‐GM076516 (EG, MD and LL).
Mechanism of Tumor Necrosis Factor alpha (TNFα) Inhibition of B0AT1 Mediated Glutamine Transport in Rat Intestinal Epithelial Cell (IEC‐6)
Authors: Talukder Jamil , Bayakly Nabil , Dawney Tonickee
Published: June 2021

Background

TNFα inhibits B0AT1‐mediated glutamine (Gln) transport by decreasing the affinity of the transporter for Gln on the brush border membrane (BBM) of IEC‐6 cells. TNFα has been shown to exert its effect on several proteins through different signaling pathways.

Aim

To identify the specific signaling pathway that TNFα activates to inhibit B0AT1‐mediated Gln transport in IEC‐6 cells.

Methods

IEC‐6 cells grown on 6‐well plates were treated with TNFα and/or specific inhibitors: Suramin, RpMBcAMP, Calphostin C, BAPTA, and Genistein for PKG, PKA, PKC, Ca2+ chelator, and ERK/JUNK, respectively for 24 h. B0AT1 function was determined by measuring [3H]‐Gln uptake. Cytosolic Ca2+ was measured using Flu 3AM. RTQ‐PCR and Western blot analyses were performed.

Results

TNFα decreased B0AT1 activity in IEC‐6 cells (7.1±0.1 nmol/mg protein/2 min in control and 2.9±0.1 in TNFα treated cells, n=4, p<0.01). Only Genistein fully blocked TNFα mediated inhibition of B0AT1 activity (6.9±0.1 nmol/mg protein/2 min). Kinetic studies demonstrated that the protective mechanism of TNFα mediated inhibition of B0AT1 by Genistein was due to the restoration of affinity of B0AT1 for Gln. Molecular studies showed that both B0AT1 mRNA and B0AT1 protein levels on BBM remained unaltered in all groups.

Conclusions

These studies revealed that TNFα inhibits B0AT1 through ERK/JUNK pathway most likely by phosphorylation in IEC‐6 cells.
Angiotensin II Inhibits P‐glycoprotein in Human Intestinal Epithelial cells
Authors: Saksena Seema , Kumar Anoop , Soni Vikas , Alrefai Waddah A , Dudeja Pradeep K , Gill Ravinder
Published: June 2021

P‐glycoprotein (P‐gp/MDR1) plays an important role in the maintenance of intestinal homeostasis. Dysregulation of Pgp function and expression have been implicated in the pathogenesis of IBD and MDR1 null mice spontaneously develop severe colitis. However, the inhibitory mechanisms of Pgp function in inflammation are not fully understood. Angiotensin II (Ang II) has been shown to exert pro‐inflammatory effects in the gut. It is increased in patients with Crohn's disease & animals with experimental colitis. Whether, Ang II directly influences Pgp is not known. Therefore, we investigated the modulation of Pgp function and expression by Ang II in human intestinal Caco2 cells. Ang II (10 nM, 60 min) inhibited Pgp activity as measured by verapamil‐sensitive 3H‐Digoxin flux (~60 %, P<0.05) with no effect at 1 nM. Also, the inhibitory effects of Ang II were observed at 60 min but not at 15 & 30 min time points. Ang II‐mediated effects on Pgp function were receptor‐mediated as the ATR1 inhibitor, losartan blocked Pgp inhibition by Ang II. Specific inhibitors of PI3 kinase, LY294002 or Akt, triciribine attenuated the effects of Ang II on Pgp activity suggesting the involvement of PI3 kinase & Akt mediated pathways. Ang II also induced Akt phosphorylation in Caco2 cells. Cell surface biotinylation studies showed that Ang II‐mediated inhibition of Pgp activity was associated with a decrease in its surface membrane expression. In conclusion, current studies demonstrate that Ang II inhibits Pgp function & membrane expression and provide novel insights into the mechanisms underlying the pro‐inflammatory role of Ang II in intestinal inflammation (Supported by NIDDK and Dept of Veteran Affairs)
Appetite regulation in desert‐adapted Spinifex hopping mice during water deprivation
Authors: Donald John Alexander , Hamid Noor Khalidah Abdul , Horvath Peter , McLeod Janet
Published: June 2021

Spinifex hopping mice, Notomys alexis, can maintain water balance without drinking. This study examined the appetite control system in hopping mice during water deprivation (WD) at five time points (2, 5, 10, 17, and 29 days) using leptin and ghrelin ELISA, and Real Time qPCR and western analysis of leptin, leptin receptor, ghrelin, ghrelin receptor, and NPY. We found that water‐deprived hopping mice showed a cyclical food intake in which hypophagia is followed by sustained food intake that is greater than water‐replete animals. The mice lost body weight primarily due to fat loss but then weight stabilised and increased as WD is prolonged (29 days). Plasma leptin decreased in parallel with fat loss but then increased again, which is probably due to leptin expression in tissues such as skeletal muscle. WD had no effect on central leptin receptor mRNA and protein. Plasma ghrelin decreased during the hypophagia but then increased significantly as food intake increased; it then decreased markedly despite a sustained food intake during the longer WD time points. As WD was prolonged, brain ghrelin mRNA expression was up‐regulated but the receptor mRNA was down‐regulated. Finally, brain NPY mRNA was up‐regulated during WD. In conclusion, central control of appetite is probably critical in hopping mice for sustaining food intake to provide substrate to generate metabolic water when free water is unavailable.
Pacific oyster mantle, gill and hemocytes express the bicarbonate‐sensing enzyme soluble adenylyl cyclase
Authors: Barron Megan Elizabeth , Roa Jinaé Nicole Bartlett , Tresguerres Martin
Published: June 2021

Soluble adenylyl cyclase (sAC) is an evolutionarily conserved CO2/pH/bicarbonate sensor involved in diverse physiological processes in vertebrates. In response to increases in bicarbonate, sAC catalyzes the formation of cAMP from ATP, which results in PKA‐dependent phosphorylation in target proteins. We have identified the Pacific oyster (Crassostrea gigas) sAC ortholog from ESTs and transcriptomic databases. Using RT‐PCR, we found sAC transcripts are present in mantle, gill and hemocytes. Using Western blot, we found Vacuolar H+‐ATPase (VHA) and carbonic anhydrase (CA) are also localized in these tissues. We propose sAC, CA and VHA are involved in sensing and counteracting elevations in CO2/pH/bicarbonate levels. Understanding the cellular mechanisms for CO2/pH/bicarbonate sensing is key in predicting biological responses to metabolic and environmental stress. For example, Ocean Acidification (OA) models project a twofold increase in dissolved CO2 (from 380ppm to 840ppm) by 2100, and sAC, CA and VHA might mediate homeostatic responses for acid/base regulation, reproduction and calcification. Support for this research was provided by a University of California Regents Graduate Fellowship.
Reverse effect of mammalian hypocalcemic cortisol in fish: cortisol stimulates Ca2+ uptake via glucocorticoid receptor‐mediated vitamin D3 metabolism
Authors: Lin Chia-Hao , Tsai I-Lun , Su Che-Hsien , Hwang Pung-Pung
Published: June 2021

Cortisol was reported to downregulate body‐fluid Ca2+ levels in mammals but was proposed to show hypercalcemic effects in teleostean fish. Fish, unlike terrestrial vertebrates, obtain Ca2+ from the environment mainly via the gills and skin rather than by dietary means, and have to regulate the Ca2+ uptake functions to cope with fluctuating Ca2+ levels in aquatic environments. Cortisol was previously found to regulate Ca2+ uptake in fish; however, the molecular mechanism behind this is largely unclear. Zebrafish were used as a model to explore this issue. Acclimation to low‐Ca2+ fresh water stimulated Ca2+ influx and expression of epithelial calcium channel (ecac), 11β‐hydroxylase and the glucocorticoid receptor (gr). Exogenous cortisol increased Ca2+influx and the expressions of ecac and hydroxysteroid 11‐beta dehydrogenase 2 (hsd11b2), but downregulated 11β‐hydroxylase and the gr with no effects on other Ca2+ transporters or the mineralocorticoid receptor (mr). Morpholino knockdown of the GR, but not the MR, was found to impair zebrafish Ca2+ uptake function by inhibiting the ecac expression. To further explore the regulatory mechanism of cortisol in Ca2+ uptake, the involvement of vitamin D3 was analyzed. Cortisol stimulated expressions of vitamin D‐25hydroxylase (cyp27a1), cyp27a1 like (cyp27a1l), 1α‐OHase (cyp27b1) at 3 dpf through GR, the first time to demonstrate the relationship between cortisol and vitamin D3 in fish. In conclusion, cortisol stimulates ecac expression to enhance Ca2+ uptake functions, and this control pathway is suggested to be mediated by the GR. Lastly, cortisol also could mediate vitamin D3 signaling to stimulate Ca2+ uptake in zebrafish.
Study of Octopamine in Bivalves
Authors: Hoque Ruma , Welsh Christopher , Ikotun Ibukun , Louis Addy Jean , Catapane Edward J. , Carroll Margaret A.
Published: June 2021

Octopamine (OA), a biogenic amine first identified in octopus, is well studied in arthropods and gastropods. Its presence and actions have rarely been reported in bivalves. Previously, we identified OA in ganglia and tissues of the oyster Crassostrea virginica (order Osteoida) and found it a cardio acceleratory agent. Here we examined OA in Mercenaria mercenaria and Mytilus edulis. Amines were measured by HPLC and spectrofluorometry. Clam and mussel heart preparations were prepared in situ by connecting ventricles to isotonic transducers. Heart rate was monitored with a Narco Systems Physiograph. Results show OA present in ng amounts, along with serotonin, dopamine and norepinephrine, in the cerebral, visceral and pedal ganglia, as well as the gill, palps, heart and foot. Mussel and clam basal heart rates averaged 12 and 8 beats/min, respectively. Superfusion of OA raised mussel heart rates but reduced clam heart rates. The actions of OA were prevented by the OA antagonist phentolamine. The study identifies OA in the nervous system and innervated organs of two additional bivalve orders, Mytilorida and Veneroida, and demonstrates OA has cardio acceleratory properties similar to C. virginica in M. edulis, but cardio inhibitory properties in M. mercenaria. This work was supported by grants 2R25GM0600309 of the Bridge Program of NIGMS, 0516041071 of NYSDOE and 0622197 of the DUE Program of NSF.
The structure of giraffe resistance arteries from muscular beds correlates with the height above ground
Authors: Aalkjaer Christian , Broegger Torbjoern , Tilenius Niklas , Broendum Emil Toft
Published: June 2021

The arterial pressure varies substantially within a giraffe. Giraffes, thus, provide ideal animals to test whether the structure of the resistance arteries reflects the transmural pressure. We dissected out small arteries from biopsies of leg muscles (ca. 80 cm above ground) and muscles from lower and upper neck (40 cm and 150 cm above the heart, neck1 and neck2, respectively from six male giraffes (Giraffa camleopardalis). The transmural pressures in the conduits at the three levels are ca. 300, 170 and 100 mmHg, respectively. The arteries were mounted in a myograph and normalized based on the passive length‐tension relationship. The normalized diameters of the arteries were 171±17 μm, 192±6 μm, 187±24 μm leg, neck1 and neck2, respectively, and did not differ, whereas the media thickness and force production (expressed as equivalent pressure) decreased significantly with the height above ground and were media thickness: 25±2 μm, 16±1 μm and 12±0 μm, leg, neck1 and neck2, respectively and equivalent pressure: 66±6 kPa, 52±6 kPa and 41±6 kPa, leg, neck1 and neck2, respectively.

We conclude that resistance artery structure and consequently their function follows the transmural pressure.
POST‐EXERCISE HYPERPNEA AND CARDIAC ASYSTOLE
Authors: Haouzi Philippe , Van De louw Andry , Bell Harold
Published: June 2021

We have recently reported that, in resting conditions, spontaneous eupneic respiration is maintained following a cardiac arrest (CA) in humans and in an animal model of ventricular fibrillation. This response deprived from any relevant role in arterial blood gas homeostasis, as circulation is abolished, still results in the production of a low level of rhythmic arterial blood pressure and in a reduction in alveolar PCO2. We are reporting here that, in a sheep model, the ventilatory control system retains during a CA the fundamental property of maintaining breathing, for 70± 41 seconds, in proportion to the level of metabolism preceding the period of asystole. As a result, ventilation persists during CA following muscle contractions but at a much higher level than at rest, despite the lack of pulmonary blood flow and cardiac output. We discuss the view that this response could be the expression of a yet unrecognised rudimentary defence mechanism aimed at limiting the consequences of an acute failure of the cardiac pump in mammals. Moreover, this “protective” response is not predictable from the traditional respiratory control feedback systems thought to be involved during exercise or its recovery.
Cyanide influence on glycolytic oscillations
Authors: Sørensen Preben Graae , Hald Bjørn Olav
Published: June 2021

Coherent glycolytic oscillations in Saccharomyces cerevisiae is a multicellular property displayed upon glucose addition to a starved cell population. The oscillations require a perturbation with cyanide, but the mechanism of cyanide influence on cell behavior remained a mystery, allthough a detailed kinetic model explain many quantitative features of the oscillations. We have studied the fate of cyanide using a cyanide electrode, and have justified experimentally the use of a cyanide electrode in the pH range of 6–7 by taking the pH into account, in a method which to our knowledge is new for studying cyanide dynamics in biological systems. During oscillations the cyanide removal was found to be aproximately 0.002mM/s. It is mainly due to reaction with extracellular acetaldehyde and evaporation of HCN. The rate constants of these processes were determined experimentally. These results have been included in a detailled mechanistic model for glycolytic oscillations, and demonstrates, that this system is an excellent model for studying metabolic dynamics in living systems.
Prolonged fasting increases purine recycling in postweaned northern elephant seals
Authors: ORGANIS JOSE GUADALUPE SONANEZ , Medina Jose Pablo Vazquez , Savin Tania Zenteno , Aguilar Andres , Crocker Daniel , Ortiz Rudy
Published: June 2021

Northern elephant seals are naturally adapted to prolonged periods (1–2 months) of absolute food and water deprivation (fasting). In terrestrial mammals, fasting stimulates ATP degradation and decreases ATP synthesis. Hypoxanthine‐guanine phosphoribosyl transferase (HGPRT) salvages ATP by recycling the purine degradation products derived from xanthine oxidase (XO) metabolism. The contributions of HGPRT to purine recycling during prolonged fasting in marine mammals are not well defined. In the present study, complete and partial HGPRT and XO cDNA sequences were obtained. Also, HGPRT and XO mRNA and protein expression was quantified in tissue from fasting elephant seals, along with the plasma levels of hypoxanthine (HX) and xanthine and activities of HGPRT and XO. Blood and tissue samples were collected from animals during the 1, 3, 5 and 7 week of their natural post‐weaning fast. The HGPRT and XO cDNA sequences encode proteins with conserved domains important for their function and regulation. HGPRT and XO mRNA and protein expression increased during the 7 wk in adipose tissue and muscle, respectively. Plasma xanthine and HX levels, and XO and HGPRT activities increased during the last two weeks of fasting. Results suggest that prolonged fasting in elephant seal pups is associated with increased capacity to recycle purines, which may contribute to enhancing the supply of ATP.