bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic hypoxia‐induced upregulation of RhoA/ROCK in adult pulmonary arteries involves ROS‐mediated covalent modification and activation of RhoA.
Authors: Negash Sewite , Gao Yuansheng , Chai Edward , Liu Jie , Tian Jia , Longo Lawrence D , Raj J. Usha
Published: June 2021

In this study, we examined the effect of chronic hypoxia (CH) on cGMP‐PKG‐mediated relaxation in pulmonary arteries. Isolated ovine intrapulmonary arteries (PAs) from ewes exposed to CH (ewes kept at 12,470 ft altitude for 110days) or matched controls (ewes kept at sea level) were preconstricted with endothelin‐1 and relaxed to 8‐Bromoguanosine 3′,5′‐cyclic monophosphate (8‐Br‐cGMP), a cell membrane‐permeable analog of cGMP, or to DETA NONOate, a stable nitric oxide donor. We found that control PAs relaxed to NO through both cGMP‐dependent and ‐independent mechanisms. In CH, NO‐mediated relaxation increased significantly, suggesting that guanylyl cyclase activity may be upregulated in these vessels. We observed minimal PKG‐mediated relaxation to 8‐Br‐cGMP both in control or CH PAs. Inhibition of ROCK with a Rho‐kinase‐specific inhibitor (Y27632), induced increased relaxation of CH PAs to cGMP, but not in control PAs, suggesting the upregulation of Rho/ROCK signaling in CH. Increased nitration of cysteine (S‐nitrosylation) and tyrosine (3‐nitrotyrosine) residues as well as a significant increase in ET‐1‐induced RhoA activation was detected in CH PAs compared to controls. These effects are mimicked by the exposure of control PAs to exogenous peroxynitrite (ONOO‐). These findings suggest that altered redox balance in chronic hypoxia and the ensuing reactive species‐mediated post‐translational modification of RhoA lead to the activation and upregulation of the Rho/ROCK constriction pathway inducing sustained pulmonary vasoconstriction.
Hypoxia Regulates Apparent PKA Activity Change in Bone Cells via Redox Potential
Authors: Zhang Yan‐Liang , Chachisvilis Mirianas
Published: June 2021

Hypoxia induced dynamic changes in the balance between PKA (protein kinase A) and its corresponding phosphatases were studied in a microfluidic environment using picosecond fluorescence spectroscopy and intramolecular fluorescence resonance energy transfer (FRET) based sensor of PKA activity. We have found that the apparent PKA activity in MC3T3 cells under hypoxia condition is regulated via redox potential‐dependent pathway rather than a cAMP‐dependent pathway. When oxygen concentration in the media was lowered due to consumption by the cells, the apparent PKA activity was increased; the lack of glucose in the media lead to a significantly lower apparent PKA activity and to a reversal of the response of apparent PKA activity to re‐oxygenation. We showed that cAMP pathway is not involved in regulating PKA activity under hypoxia condition since changes in cAMP level in the cells were not detected using a cAMP FRET sensor. We have shown that the rate of oxygen consumption, as detected using a proporphyrin based phosphorescent oxygen sensitive probe, correlates with the rate of the change in apparent PKA activity, and so does the rate of change in NADH concentration. We suggest that the apparent PKA activity change in cells under hypoxia is due to hypoxia regulated response of phosphatases acting on substrate of the PKA. This research was supported in part by the NIH grant HL086943.
Formation of signaling‐related oxidative base modifications in the hypoxic response element (HRE) of the VEGF gene in hypoxic pulmonary artery endothelial cells (PAECs) is associated with chromatin remodeling
Authors: Ruchko Mykhaylo V. , Gorodnya Olena , Pastukh Viktor M. , Gillespie Mark N.
Published: June 2021

In PAECs, hypoxia causes oxidative DNA modifications in the HRE of the hypoxia‐inducible VEGF gene (FASEB J., 19:387–394, 2005). Because introduction of a base oxidation product mimicking the effect of hypoxia facilitated both transcription factor binding to the VEGF HRE and reporter gene expression, we proposed that hypoxia‐induced base modifications regulate VEGF expression. If this hypothesis is valid, then base oxidation products should be detected in the VEGF HRE upon transcriptional activation. Accordingly, DNA released from normoxic and hypoxic PAECs by micrococcal nuclease treatment was separated into transcriptionally‐active mono‐ and di‐nucleosomes and inactive multi‐nucleosomes. Southern blot analysis indicated that hypoxia‐induced VEGF mRNA expression was linked to redistribution of the HRE from multi‐nucleosomes to mono‐ and di‐nucleosome fractions. A simple PCR method showed that hypoxia‐induced base oxidation products in the VEGF HRE accumulated exclusively in the mono‐nucleosomal fraction. When redistribution of the VEGF HRE into mono‐nucleosomes was suppressed by a histone deacetylase inhibitor, formation of base oxidation products in the sequence was prevented. These findings suggest that hypoxia‐induced oxidative modifications targeted to specific nucleotides in the VEGF HRE are associated with chromatin remodeling and transcriptional activation.

Supported by NIH.
TGF β‐1 mediated increase in Nox‐4 expression enhances hypoxic pulmonary vasoconstriction in bovine pulmonary arteries
Authors: Ahmad Mansoor , Zhao Xiangmin , Kelly Melissa R. , Kaminski Pawel M. , Wolin Michael S.
Published: June 2021

Hypoxic pulmonary vasoconstriction (HPV) in de‐endothelialized bovine pulmonary arteries (BPA) is potentially mediated through lowering of superoxide derived peroxide and removal of its basal relaxing effect. Simultaneous force and peroxide measurements showed decreased peroxide levels preceding HPV. Peroxide scavenging with 0.1mM ebselen increased 25mM KCl force and attenuated HPV. BPA pretreated with copper chelator 10mM diethyldithiocarbamate (DETCA), for inactivating Cu Zn SOD, increased 5μM lucigenin‐detectable superoxide and decreased horseradish peroxidase‐luminol detectable peroxide. DETCA also increased 20mM KCl force and attenuated HPV. 48 hr culture of BPA with 10nM Transforming growth factor β‐1 (TGF β‐1) increased NAD(P)H oxidase‐4 (Nox‐4) expression along with superoxide and peroxide levels. 10μM plumbagin (Nox‐4 inhibitor) reversed the increase in superoxide and peroxide with TGF β‐1, however 100μM apocynin (Nox‐2 inhibitor) did not reverse the increased superoxide level. Also TGF β‐1 decreased 20mM KCl force and increased HPV in BPA. Thus, TGF β‐1 mediated increase in Nox‐4 resulted in higher levels of superoxide derived peroxide in BPA which increased HPV.

NIH grants (HL31069/HL66331).
Effect of cardiovascular exercise on CRP levels in previously sedentary older men and women
Authors: Pence Brandt D. , Vieira Victoria J. , Baynard Tracy , Keylock K. Todd , Lowder Thomas W. , Woods Jeffrey A.
Published: June 2021

Purpose:

To assess changes in serum C‐reactive protein (CRP) with 10 mo of cardiovascular exercise training in sedentary 65–80 yr old subjects.

Background:

CRP is a clinical marker of systemic inflammation, and a chronic inflammatory state has been implicated in the pathogenesis of diseases including type 2 diabetes (T2D) and coronary heart disease (CHD), which are often found in aged persons.

Methods:

Sedentary male and female participants (n=120, 70 ± 1 years) were randomly assigned to 2 groups: a cardiovascular exercise group (EX; n = 61) and a flexibility/balance control group (CON; n = 59). EX subjects completed 10 mo of training (3x/wk walking, cycling, elliptical) at 65–70% VO2 max. CON subjects completed flexibility exercises 2x/wk to eliminate attention bias. Serum CRP was analyzed via an enzyme‐linked immunosorbant assay.

Results:

The EX group showed reduced CRP levels over the course of the intervention (3.66 ± 0.34 vs. 3.22 ± 0.36 mg·L−1; P < 0.01), with no reduction in CRP levels in the CON group (3.21 ± 0.28 vs. 3.31 ± 0.28 mg·L−1).

Conclusion:

Ten months of endurance exercise training reduced serum CRP in older subjects exhibiting elevated CRP levels. The reduction in CRP seen with cardiovascular exercise training suggests an overall decrease in systemic inflammation. This may contribute to the reduction in the risk of diseases such as T2D and CHD with exercise.

Supported by R01 AG‐18861 to J.A. Woods
Effect of whey protein or carbohydrate supplementation on markers of muscle damage, muscular dysfunction and leg soreness
Authors: Witard Oliver Charles , Jackman Sarah R , Jeukendrup Asker E , Tipton Kevin D
Published: June 2021

This study aimed to determine the impact of whey protein (WP) or carbohydrate (CHO) supplementation on recovery from exercise‐induced muscle damage. In a parallel, placebo‐controlled, single‐blind experimental trial, untrained males were randomly assigned to a WP (n=12), CHO (n=12) or placebo (n=12) supplementary group. Muscle damage was elicited by implementing an eccentric unilateral leg extensor protocol (15×10 repetitions at 170% peak isokinetic torque) using an isokinetic dynamometer. Diet was strictly controlled throughout the trial. Measures of muscle function, muscle damage, leg soreness and inflammation were taken pre‐ and post‐ (1, 8, 24, 48 and 72h) exercise. Maximal isometric strength (MIS) decreased by ~32% at 48h post‐ (662.3±32.2N) compared to pre‐exercise (451.2±38.9N) (P<0.01). Ratings of leg soreness peaked 24h following exercise. No effect of supplement was observed on post‐exercise plasma creatine kinase (CK) or myoglobin responses, MIS, visual analogue scale ratings or leg volume (P>0.05). Measures of muscle function and CK showed no correlation (r=−0.211) (P=0.402). These data suggest the chosen experimental model successfully increased muscular dysfunction, leg soreness and putative markers of muscle damage. However, supplementation with 40g/d WP or CHO failed to ameliorate these symptoms of muscle damage under conditions of dietary control.

Protarmor 907 LSI whey protein was supplied by Armor Proteines, Saint‐Brice‐en‐Cogles‐FRANCE
A new device to estimate energy expenditure during uphill and downhill walking by accelerometry and barometry
Authors: Yamazaki Toshiaki , Gen‐no Hirokazu , Nose Hiroshi
Published: June 2021

We developed a new device to estimate energy expenditure during uphill and downhill walking. First, we measured oxygen consumption rate (VO2, respiratory gas analysis) and 3 dimensional accelerations in 42 healthy people (63±7(SD) yr) during graded walking on a treadmill while slope was varied from −15% to +15%. They walked for 3 minutes at subjectively slow, moderate, fast and fastest speeds on flat and uphill settings and walked at ∼3, 4 and 5km/h for downhill settings. We determined a regression equation to estimate VO2 from the sum of total impulse (Itotal, N · min), theoretical potential energy gain (Pg, m/min · kg) and loss (Pl, m/min · kg) for last one minute of each trial, as VO2=0.047Itotal+0.0014Pg+0.0006Pl, where Itotal was determined from a square root of summed accelerations of each direction, and Pg or Pl from a product of body weight and theoretical height changes. Second, to validate the precision of the equation, we measured VO2, Itotal and barometric pressure in 11 of 42 subjects walking in the field. The height change was estimated from change in barometric pressure. A regression equation between estimated VO2 (y) and measured VO2 (x) in the range of 0.37∼2.56 l/min was y=0.98x+0.04 (r=0.88, P<0.001) with mean difference of 0.01±0.23 (SD) in Brand‐Altman plot. Thus, energy expenditure during uphill and downhill walking was precisely estimated with a device composed of accelerometers and a barometer.
Life or death in the fast lane: dolphin mother‐calf separation during tuna purse‐seine sets
Authors: Edwards Elizabeth F.
Published: June 2021

Dolphin stocks in the eastern tropical Pacific Ocean, reduced 70–80% due to fishery‐related mortality 1960–1990, are not recovering despite near‐zero fishery mortality since 1991. Because mortality records show that 75–95% of killed lactating females are not killed with a nursing calf, the fishing process appears to separate mothers and calves, so that unobserved mortality of calves may be important to the lack of stock recovery. A suite of studies investigated this question from a number of perspectives, including review of physiological and behavioral development in dolphin calves, mathematical analysis of dolphin drafting hydrodynamics, and empirical studies of swim development in dolphin calves from birth through two years. In toto, these studies show that the sustained high‐speed swimming and accompanying aerial respirations characteristic of dolphins evading purse‐seine sets creates conditions under which dolphin calves will have great difficulty sustaining a drafting relationship with the mother, and that developmental limits are likely to prevent calves from maintaining an association with the mother while swimming independently. Effects are strongly age‐related, with youngest calves at greatest risk for separation.
Endothelial cilia are mechanosensory organelles
Authors: Nauli Surya M , Kawanabe Yoshifumi , Kaminski John J , Pearce William J , Ingber Donald E , Zhou Jing
Published: June 2021

BACKGROUND

When challenged with extracellular fluid‐shear stress, vascular endothelial cells are known to release nitric oxide (NO), an important vasodilator. We here show that this mechanosensing signal transduction depends on apical membrane organelles, termed cilia.

METHODS AND RESULTS

Cells with Pkd1null/null or Tg737orpk/orpk mutation encoded for polycystin‐1 or polaris, respectively, are unable to transmit extracellular shear stress into intracellular calcium signaling and biochemical NO synthesis. Cytosolic calcium and NO recordings further show that fluid‐shear sensing is a cilia‐specific mechanism, as other mechanical or pharmacological stimulation does not abolish calcium and NO signaling in polycystin‐1 and polaris mutant endothelial cells. Polycystin‐1 localized in the basal body of Tg737orpk/orpk endothelial cells is insufficient for a fluid‐shear stress response.

CONCLUSION

We demonstrate for the first time that the ability of endothelial cells to sense a low range of fluid shear depends on cilia and that cilia are compartments required for proper localization and function of the mechanosensitive polycystin‐1 molecule.
Enhancement of ciliary beat frequency induced by [Cl−]i decrease in rat distal airway ciliary cells
Authors: Takemura Yoshizumi , Iwasaki Yoshinobu , Marunaka Yoshinori , Eaton Douglas C. , Nakahari Takashi
Published: June 2021

Regulation of ciliary beat frequency (CBF) plays a key role in the maintenance of mucociliary clearance, which is a host defense mechanism of lungs. A previous report has shown that cell shrinkage enhances terbutaline (Tb, a β2‐adrenergic agonist)‐stimulated CBF. As an isosmotic cell shrinkage causes a decrease in the intracellular chloride ion concentration ([Cl−]i), we examined the effects of an [Cl−]i decrease on the CBF enhancement. [Material and method] Ciliary cells in the distal airway of rat were isolated by an elastase treatment, and their CBFs were measured using a high speed camera. [Results] The application of Cl−‐free solution or bumetanide, which decreased the [Cl−]i, increased CBF and enhanced the Tb‐stimulated CBF. The application of 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB), which increased the [Cl−]i, decreased CBF, however it did not suppress the Tb‐stimulated CBF. An inhibitor of PKA (H89) decreased CBF to 40% and eliminated the enhancement of CBF induced by the Cl−‐free solution and the Tb (10 μM). [Conclusion] A decrease in [Cl−]i enhanced CBF by modulating PKA signalings.
Protein Kinase C Activity and Cell Proliferation in Polycystic Kidney Disease
Authors: von der Heiden Jennifer , Siroky Brian , Gooz Monika , Komlosi Peter , Bell Phillip Darwin
Published: June 2021

Polycystic kidney disease is associated with the loss of the structure/function of the primary cilia. Cilia regulate intracellular calcium concentration ([Ca2+]i), with the loss of cilia leading to an elevation in [Ca2+]i. These studies examine whether this increase in [Ca2+]i would lead to protein kinase C (PKC) activation and whether activated PKC would contribute to cell proliferation in polycystic kidney disease. Studies were performed in cilia (‐) and cilia (+) collecting duct cell lines, derived from the Tg737orpk mouse. Cilia (‐) cells compared to cilia (+) cells displayed reduced cytosolic PKC content. There was also increased translocation of PKC to the plasma membrane in cilia (‐) cells, as assessed with a α‐PKC antibody and confocal microscopy. In other studies, cilia (‐) cells proliferated at a much higher rate than cilia (+) cells. Chronic treatment with 10 μM BAPTA‐AM, a Ca2+ chelating agent, reduced the proliferation rate in cilia (‐) cells to a level comparable to cilia (+) cells. Importantly, inhibition of α‐PKC with Ro‐32‐0432, also abolished increased proliferation in cilia (‐) cells compared to cilia (+) cells. Thus, we suggest that there is Ca2+‐dependent increased α‐PKC translocation in cilia (‐) cells and that elevated PKC activity contributes to the hyper‐proliferation and possibly cyst formation in this model of polycystic kidney disease.
The Role of Focal Adhesion Kinase in Sphingosine‐1‐Phosphate Induced Endothelial Barrier Enhancement
Authors: Thennes Tracy Lynn , Konstantoulaki Maria , Mehta Dolly
Published: June 2021

Dynamic interactions between adherens junctions and cell‐matrix adhesions mediated by actin cytoskeleton regulate endothelial barrier function. Barrier‐enhancing agonist, sphingosine‐1‐phosphate (S1P), by binding its S1P receptor1 (S1P‐1) on the endothelial cell surface enhance endothelial monolayer integrity by Rac1‐mediated stabilization of intercellular junctions and by promoting cell‐matrix adhesions at focal adhesions complexes (FACs). Focal adhesion kinase (FAK) regulates the cell‐matrix adhesive contact at focal adhesion sites. We address the possibility that S1P via FAK‐induces Rac1 activation which in turn signals lamellopodia formation to promote cell‐cell interactions and endothelial barrier enhancement. We show that S1P induces FAK activation, as evidenced by phosphorylation on tyrosine residues 397 and 576 in endothelial cells, in association with enhancement of endothelial barrier function. In YFP‐actin mutant transducing cells S1P induced robust lamellopodia formation, consistent with the role of Rac1 in strengthening intercellular junctions. Inhibition of FAK by transduction of dominant negative FAK mutant (FRNK) markedly suppressed basal Rac1 activity. FRNK expression also increased basal endothelial permeability as measured by determining transendothelial monolayer resistance and S1P could not restored it to the level seen in control cells. These findings suggest that S1P induced FAK activation may play an important role in Rac1 mediated endothelial barrier enhancement.
Lung platelet‐endothelial interactions in high tidal volume ventilation
Authors: Yiming Maimaiti T. , Bhattacharya Sunita
Published: June 2021

Platelets amplify endothelial P‐selectin expression in high tidal volume ventilation (AJRCMB, 2005, 33:549‐54) but mechanisms underlying platelet‐endothelial interactions are not known. We considered the roles of the platelet inflammatory proteins, glycoprotein 1b alpha (GP1b‐α), vWf and P‐selectin. We recovered lung endothelial cells (FLEC) from freshly excised (control), or isolated, blood‐perfused rat lungs that we ventilated at tidal volume of 12 ml/kg for 2h (HV). Confocal microscopy of immunofluorescently labelled FLEC revealed that GP1b‐α and vWf were weakly expressed for control, but strongly expressed and colocalized for HV. Lung perfusion with the P‐selectin function‐blocking mAb, RMP‐1 markedly inhibited the HV‐induced vWf expression (P<0.05). In contrast to WT, HV failed to increase vWf expression in FLEC from P‐selectin knockout (P‐sel−/−) mice. This inhibitory effect was rescued by perfusing P‐sel−/− lungs with WT blood. We conclude that in HV, platelets transfer GP1b‐αto lung EC and that platelet P‐selectin determines EC vWf expression. Lung recruitment of inflammatory cells might result from platelet‐EC transfer of critical inflammatory receptors (Support: HL54157).
Expression of focimatrix correlates with expression of enzymes for estradiol and progesterone synthesis in ovarian follicles
Authors: Rodgers Raymond Joseph , Irving‐Rodgers Helen Frances
Published: June 2021

Focimatrix is basal lamina matrix containing collagens, laminin, nidogens and perlecan and is deposited as aggregates, not sheets, between, and not around, the epithelial granulosa cells of ovarian follicles [Irving‐Rodgers et al. (2004) Matrix Biol 23, 207–217]. Focimatrix is developmentally regulated and its function is not known. We therefore measured expression of its components in dominant and subordinate bovine ovarian follicles relative to other indices of granulosa cell maturation. Pairs of ovaries were collected at an abattoir from cycling, non‐pregnant cows (n = 30) and the three largest follicles were harvested per cow (8–18 mm). Only healthy follicles were examined (n = 44) and granulosa cells were isolated for RNA extraction and qRT‐PCR analyses of FSHR, LHR, INHB, CYP11A1, CYP19, COLIVA1, LAMB2, HSPG2, NID1 and NID2. Correlation analysis found that the expression of key enzymes in the synthesis of progesterone and estradiol, CYP11A1 and CYP19, were highly correlated (up to P<0.0001) with expression levels of the focimatrix components COLIVA1, LAMB2 and HSPG2. These results suggest a causal relationship between focimatrix production by granulosa cells and estradiol and progesterone synthesis. Since focimatrix precedes production of steroid hormones during follicle development we conclude that focimatrix could regulate steroid hormone synthesis. Funded by NHMRC.
Boric acid is a modulator of stored calcium release in DU‐145 prostate cancer cells
Authors: Henderson Kimberly , Eckhert Curtis D
Published: June 2021

We have reported that both incidence and mortality of prostate cancer (PC) are decreased in a dose dependent manner by boron in diet (Oncology Rep 2004) and drinking water (Cancer Causes Control 2007). Boric acid (BA) inhibits PC cell proliferation (Cancer Lt 2004), binds to NAD+ and inhibits ADP ribosyl cyclase (J Mass Spec 2003,2004; J Chromatography 2006). Our present objective was to determine BA's effect on Ca2+ store release in DU‐145 cells. Endoplasmic reticulum (ER) Ca2+ was labeled with Rhod 2 and Ca2+ release was monitored using a Zeiss LSM 5 Pascal laser confocal microscope. BA inhibited Ca2+ release in response to 10 ìM cyclopiazonic acid (SERCA inhibitor) and 20 mM caffeine (ryanodine channel agonist) in a dose dependent manner beginning at 0.1 ìM BA. BA inhibited Ca2+ release in response to 4‐chloro‐m‐cresol in a dose dependent manner from 0.1 ìM BA to near complete inhibition at 10 ìM BA. Inhibition of caffeine induced Ca2+ release required >10 fold higher concentrations BA (150 ìM) in PWR1E non‐tumor cells than DU‐145 tumor cells. Our results demonstrate that BA acts as a ryanodine receptor antagonist and suggests boron's chemopreventative effect results from its ability to dampen ryanodine channel Ca2+ signaling. Funding: DAMD 17‐03‐1‐0067 and UC TSR&TP.
Omeprazole abrogates cell protection in an in vitro model of infectious gastritis
Authors: Blass Amy L. , Kohler Jonathan E. , Liu Jingjing , Mathew Jeff , Tai Kaniza , Kelly Edward , Soybe David I.
Published: June 2021

Aim:

Monochloramine (NH2Cl) is a thiol oxidant produced in H. pylori gastritis. Omeprazole (OME) is a proton pump inhibitor, and a thiol oxidant, used to treat gastritis. Our aim was to determine if OME influences cell injury by NH2Cl, and to test if effects of these thiol oxidants are mediated by changes in [Ca2+]i and [Zn2+]i.

Methods:

Isolated rabbit gastric glands were incubated with doses of NH2Cl for 3 hours, and cell viability was measured with Calcein‐AM. Chelators (BAPTA or TPEN) and a thiol reducing agent (DTT) were added, with or without OME. Results were normalized to Ringers controls.

Results:

NH2Cl caused cell death at all concentrations. DTT protected cells from NH2Cl‐induced damage. The promiscuous chelator BAPTA gave no protection. However, the zinc‐specific chelator TPEN protected completely. When cells were co‐incubated with OME, cell death occurred even in the absence of NH2Cl. Addition of TPEN to these cells was not protective despite high [Zn2+]i, but addition of DTT again prevented cell death. Conclusion: Chelation of intracellular Zn2+, but not Ca2+, protects cells exposed to NH2Cl. However, OME, commonly used to treat H. pylori gastritis, appears to cause zinc‐independent cell death. This can be reversed by the thiol‐reducing agent DTT, suggesting that OME acts downstream of Zn2+‐mediated injury. Funding: NIH R01DK069929‐03
Novel small molecules reverse myosin light chain kinase (MLCK) isoform 1 translocation and barrier dysfunction induced by tumor necrosis factor (TNF)
Authors: Graham W. Vallen , Marchiando Amanda M. , Wang Yingmin , Ostrov David A. , Turner Jerrold R.
Published: June 2021

MLCK is central to TNF‐induced intestinal epithelial barrier dysfunction in vitro and in vivo. MLCK is expressed as two splice‐variants, MLCK1 and MLCK2, distinguished by a short insertion in MLCK1. MLCK1 knockdown increases epithelial barrier function in vitro. We therefore hypothesized that MLCK1 might specifically regulate the perijunctional actomyosin ring (PAMR) during TNF‐induced barrier dysfunction.

TNF‐treated Caco‐2 monolayers demonstrated a progressive increase in PAMR‐associated MLCK1 within 30 min, 35.7±0.1% (P<0.05), that peaked at 4 hr, 118.8±0.1% (P<0.001), and correlated directly with TNF‐induced barrier loss (r=0.9). Structural modeling showed that the MLCK1 insertion created a binding pocket. In silico screening identified candidate small molecules targeting this pocket, of which, one molecule, SMKI2, bound the pocket with a predicted ΔG of −92 kcal/mole. 59.9±0.2% (P<0.001) of MLCK1 was displaced from the PAMR by 250 μM SMKI2. In contrast, enzymatic MLCK inhibition caused no MLCK1 displacement. However, like enzymatic inhibition, SMKI2 corrected TNF‐induced barrier loss in a dose‐dependent fashion.

These data provide insight into the mechanism of TNF‐induced MLCK activation and PAMR myosin phosphorylation. Inhibition of MLCK1 targeting may be an important means of correcting barrier dysfunction without the potential toxicities of enzymatic MLCK inhibition.

Supported by NIH
Acute Treatment of Isolated Colon with The Hydrogen Peroxide Scavenger, Catalase, Alleviates Ion Transport Dysfunction in Murine DSS Colitis
Authors: McCole Declan F , Barrett Kim E
Published: June 2021

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) contribute to epithelial dysfunction in inflammatory bowel disease (IBD). This study investigated the role of H2O2 in colonic ion transport defects in the murine DSS colitis model.

Colitis was induced by administering DSS (4%) in drinking water for 5 days followed by 3 days on normal H2O. Distal colon from control and DSS mice was stripped of underlying smooth muscle and mounted in Ussing chambers. Tissues were bilaterally treated with the H2O2 scavenger, catalase (50, 100 or 300 U/ml), for 30 min prior to measuring short‐circuit current (Isc) responses to the cAMP agonist, forskolin (20 μM), or the Ca2+‐dependent muscarinic agonist, carbachol (CCh, 300 μM). Catalase (100 U/ml) treatment of tissues from DSS mice increased baseline Isc above that of untreated tissues (p<0.01; n=7) without affecting baseline Isc in control mouse colon. Catalase significantly restored the blunted Isc responses to forskolin (p<0.05; n=7) and CCh (p<0.05; n=7) in DSS mouse colon but had no effect in control mouse colon (n=4). Incubation of inflamed colon with catalase also abolished H2O2 release into bathing media, as measured by a colorimetric H2O2 assay (n=3).

In summary, these data indicate that ongoing production of H2O2 in colitis contributes to the reduced capacity for active ion transport in inflamed colon.

Supported by the Crohn's and Colitis Foundation of America and NIH.
Ion transport deficits observed during colitis are associated with functional inhibition, but not decreased expression, of IKCa channels ‐ possible involvement of PKC
Authors: Hirota Christina L , McKay Derek M , MacNaughton Wallace K
Published: June 2021

Adequate hydration of the intestinal epithelial surface is essential for gut function and is regulated by active ion transport. We have shown in a dextran sodium sulfate (DSS) model of colitis that colonic secretory responses to bethanechol are abolished and responses to histamine, thapsigargin, cyclopiazonic acid, and the calcium ionophore A23187 are severely attenuated. We hypothesized that this could be due to decreased expression and/or activity of intermediate‐conductance Ca2+‐activated K+ channels (KCNN4/IKCa). Analysis of mRNA extracted from colonic crypts showed no transcriptional differences in KCNN4. Western blotting revealed no changes in IKCa protein expression, although calmodulin was increased in crypts from DSS‐treated mice. Direct activation of IKCa channels with DCEBIO produced a highly diminished ion transport response in DSS compared to controls. This apparent functional inhibition of IKCa channels during DSS colitis could be due to aberrant Ca2+ signaling, possibly via protein kinase C (PKC). Indeed, ion transport responses in control tissue are sensitive to non‐selective PKC inhibition; also, PKC activation with phorbol 12‐myristate 13‐acetate led to increased ion transport in control colonic tissue that was greatly reduced in DSS tissue. This hyposecretory phenotype may represent a strategy to counter the malabsorptive diarrhea associated with colitis. Funded by the CCFC.
The Effect of IL‐6 in the Anemia of Inflammation
Authors: Pastuszka Martha Katherine , Yu Alice , Ganz Tomas , Rivera Seth
Published: June 2021

Anemia of inflammation (AI anemia of chronic disease), results from a large variety of inflammatory conditions. Hepcidin, the principal systemic iron regulatory hormone, is increased during inflammation and limits the availability of iron for erythropoiesis. IL‐6, an inflammatory cytokine, has been shown to be necessary for the elevation of hepcidin during acute inflammation (Nemeth et al, Blood, 2004). However, it has been shown that IL‐1 can also induce hepcidin in vitro (Lee et al, PNAS, 2005). To explore whether IL‐6 is necessary for the development of AI we used a mouse model of AI induced by a single IP injection of complete Freund's adjuvant in IL‐6 KO and WT mice. Hemoglobin concentration decreased to the same extent in WT and KO mice. Hepcidin increased in WT mice but did not change in KO mice. Mean corpuscular volume (MCV) decreased in WT mice but increased in KO mice. Hepatic serum amyloid A mRNA, a marker of hepatic IL‐6 responsiveness, increased in WT but not IL‐6 KO mice indicating that the anemia was not a result of IL‐6‐like cytokines. IL‐6 is not necessary to develop AI. However, IL‐6 does effect the handling of iron as evidenced by the difference in cell size and hepcidin production.
Maternal sleep deprivation exposure and the consequences in offspring
Authors: Calegare Bruno Frederico Aguilar , Fernandes Leandro , Tufik Sergio , D’Almeida Vânia
Published: June 2021

Growing interest has been driven by the discovery of a tight relationship between prenatal manipulations and development of short‐ and long‐term health disorders. Our objective was to analyze developmental programming effects of maternal sleep deprivation on offspring biometric and behavioral parameters. Pregnant female Swiss mice aged 3 months were distributed into 3 groups: females deprived of sleep by gentle handling (for 6 hours) on 24 hours of pregnancy (GH24) and on 48 hours of pregnancy (GH48); and a last group which could sleep normally (CT). The offspring was weighted until the 3rd month of age, when they were tested at activity box and elevated plus maze (EPM). The offspring weight was different at birth, and differences were observed until the end of the 1st month, when they were weaned and the differences disappear. At EPM apparatus the GH24 group presented a decrease of time spent in the closed arms when compared to GH48 group, and GH48 group defecated less when compared to CT group. This data suggest that sleep deprivation of early mothers lead to physiologic alterations from birth to wean, and behavioral alterations were still observed in adulthood. Support: FAPESP, CNPq and AFIP.
Methylated N‐(4‐N,N‐dimethylaminobenzyl) chitosan enhanced epithelial permeability
Authors: opanasopit Praneet , Kowaprodit Jariya , Ngawhiranpat tanasait , Rojanarata Theerasak , Ruktanonchai Uracha , Sajomsang Warayuth
Published: June 2021

The purpose of this study was to investigate the modified CS, methylated N‐(4‐N,N‐dimethylaminobenzyl) chitosan (TM‐Bz‐CS), on the transepithelial electrical resistance (TEER) and permeability of Caco‐2 cells monolayer, using FITC‐dextran (FD‐4). TM‐Bz‐CS was prepared by substitultion of trimethylaminobenzyl units on the CS backbone and N‐trimethyl quaternization. These studies showed that TM‐Bz‐CS appeared to increase cell permeability in dose‐dependent manner and caused relatively reversible effects only at the lower doses (0.001−0.01 % w/v) on epithelial function. Both benzyl substitutions (TM‐Bz17‐CS and TM‐Bz42‐CS) were not significantly different in TEER values since they showed rapidly reduction in TEER values within 20 min after starting of the experiment. Cytotoxicity of both TM‐Bz‐CSs were concentration dependent and higher than CS. In conclusion, these studies demonstrated that TM‐Bz‐CS appear to increase cell permeability in the dose dependent and cause relatively reversible effects on epithelial function. Amount of TM‐Bz‐CS influenced on the permeation enhancing effects. Moreover, TM‐Bz‐CS was safety for using as absorption enhancers. This research was supported by Royal Golden Jubilee scholarship, Thailand Research Fund, Thailand.
Functional expression of human organic cation transporter 3 in renal cancer cells
Authors: Shnitsar Volodymyr , Koepsell Hermann , Burckhardt Gerhard , Hagos Yohannes
Published: June 2021

Renal cancer cell (RCC) carcinoma is usually chemoresistant. This chemoresistance would be overcome when a cytostatic is applied for which the RCC possess an uptake transporter. Therefore, in the present study we investigated the expression of solute carrier (SLC) transporters in different RCC samples and their ability to interact with chemotherapeutical drugs. We tested five RCC cancer lines as well as normal and tumor tissue for expression of 30 different SLC by RT‐PCR and TaqMan real‐time PCR. In two of five RCC lines, A498 and 7860, we observed a highly significant expression of SLC22A3 (hOCT3). The uptake of organic cation [3H] MPP (4‐methyl‐pyridinium iodide) into these cells and also into hOCT3 stably transfected CHO cells was highly inhibited by irinotekan and vincristine. The Ki values calculated from Dixon plots for irinotekan and vincristine are 1.72±0.45 μM and 17±4.81 μM, respectively. Cytotoxic activities of the selected drugs were tested by the MTT assay on CHO‐OCT3, A498 and ACHN cell lines. The growth of CHO‐OCT3 was inhibited by 20% more with irinotekan and by 50% more with vincristine compared to non transfected CHO cells. Similar results were obtained for A498 and ACHN cells. Thus, our data support the hypothesis that the sensitivity of tumor cells to chemotherapeutical treatment depends on the expression of transporter proteins mediating specific drug accumulation into the target cell.
Expression Level of Canonical Transient Receptor Potential (TRPC) Channels is Increased in the Adrenal Medulla of Ossabaw Miniature Pigs Manifesting the Metabolic Syndrome
Authors: Hu Guoqing , Neeb Zachary P , Sturek Michael , Obukhov Alexander G
Published: June 2021

The objective of this study was to investigate whether pig adrenal chromaffin cells (ACC) express G q/11‐protein coupled receptors (GPCR) and whether excess kcal atherogenic diet that elicits metabolic syndrome (MetS) in Ossabaw pigs increases TRPC expression in the adrenal medulla (AM). TRPCs are Ca2+‐permeable cation channels that are activated by agonists of GPCR. TRPC channels mediate circulating hormone‐stimulated, voltage‐independent exocytosis of catecholamines from ACC. Higher levels of plasma epinephrine are found in MetS animals vs. lean control animals, suggesting enhanced exocytosis from AM. We used molecular biological, biochemical and fluorescence imaging methods to study GPCRs and TRPCs in ACC isolated from AM of the lean and MetS pigs. Using fluorescence imaging, we demonstrated that angiotensin II, muscarinic, and histamine GPCRs are expressed in porcine ACC, and that the receptors’ activation stimulates Ca2+/Mn2+ influx. We cloned pig TRPC1 and TRPC3–7 channels. We detected transcripts of TRPC1, TRPC5 and TRPC6 in the total RNA isolated from AM. The expression of TRPC1, TRPC5 and TRPC6 was significantly higher in AM from MetS vs. lean pigs. We conclude that an abnormal up‐regulation of adrenal TRPCs in MetS may underlie the elevated levels of plasma epinephrine. Support: NIH RR013223 and HL062552 to MS and HL083381 to AGO.
Functional and electrophysiological identification of large conductance voltage‐ and Ca2+‐activated K+ (BK) channels in human urinary bladder smooth muscle
Authors: Hristov Kiril L. , Cui Xiangli , Kellett Whitney F. , Rovner Eric S. , Petkov Georgi V.
Published: June 2021

The large conductance voltage‐ and Ca2+‐activated K+ (BK or KCa1.1) channel is a key player in controlling excitability and contractility of animal urinary bladder smooth muscle (UBSM), however its role in human UBSM is unknown. Here, using the BK channel specific inhibitor‐iberiotoxin, functional studies and the perforated patch‐clamp technique, we identified the BK channel in human UBSM. In isolated UBSM strips iberiotoxin increased spontaneous phasic contraction amplitude and force (n=10). In isolated UBSM cells, voltage‐step depolarizations caused large K+ currents (∼1 nA; n=15). About 80% of these K+ currents were inhibited by iberiotoxin. In the voltage range of −40 to +40 mV, these cells also revealed spontaneous transient outward currents (STOCs). STOCs amplitude and frequency increased with increasing the holding potential toward positive values (n=20). STOCs were inhibited by iberiotoxin (n=4). In the presence of ryanodine, when the STOCs were blocked, single channels with large single channel conductance (120.6 pS) were observed (n=10). Their amplitude and open probability were voltage‐dependent. In current‐clamp mode, iberiotoxin caused membrane potential depolarization (n=4). The data reveal that the BK channel is the major voltage‐dependent K+ channel expressed in human UBSM cells that plays a key role in determining cell excitability and contractility. Supported by DK070909 to G.V.P.
High‐ and low‐capacity taurine transport across the luminal membrane of the small intestinal epithelium
Authors: Anderson Catriona M.H. , Ganapathy Vadivel , Thwaites David T.
Published: June 2021

Taurine is a “semi‐essential” beta‐amino acid; yet it is not clear which transporter(s) mediates taurine uptake across the small intestinal luminal membrane. The three intestinal beta‐amino acid transporters [taurine transporter TauT (SLC6A6), proton‐coupled amino acid transporter PAT1 (SLC36A1), and ATB0,+ transporter (SLC6A14)] were expressed in Xenopus laevis oocytes, and radiolabeled β‐alanine or taurine uptake characterized. TauT and PAT1 both transport taurine and β‐alanine whereas ATB0,+ transports only β‐alanine. To determine the relative contribution of each carrier to small intestinal transport, β‐alanine and taurine uptakes were measured across the apical membrane of human intestinal Caco‐2 cell monolayers. Use of selective inhibitors and ion substitutions identified roles for PAT1 and TauT (but not ATB0,+) in small intestinal uptake of these beta‐amino acids (ATB0,+ function may be more important in the colon). Under physiological conditions (apical pH 6.5, presence of Na+), the relative contributions of PAT1 and TauT to taurine uptake were dependent upon the taurine concentration. The high‐capacity, low‐affinity PAT1 may provide the major route for small intestinal taurine absorption from taurine‐rich foods whereas the low‐capacity, high‐affinity TauT may scavenge small quantities of taurine from the lumen. Supported by The Wellcome Trust (grant number 078640/Z/05/Z).
Comparison of contractile mechanisms of sphingosylphosphorylcholine and sphingosine‐1‐phosphate in rabbit coronary artery
Authors: Choi Soo‐Kyoung , Ahn Duck‐Sun , Lee Young‐Ho
Published: June 2021

We investigated the difference of patterns and specific mechanisms between SPC‐ and S1P‐induced contraction in rabbit coronary artery. Contractile responses in normal and permeabilized strips, changes in [Ca2+]i, and phosphorylation of MYPT1 were measured. SPC induced a gradual and sustained contraction. S1P induced initial transient and secondary sustained force. In isolated single cells, SPC induced a slight increase in [Ca2+]i but S1P significantly increased [Ca2+]i. In the absence of extracellular Ca2+, S1P‐induced [Ca2+]i increase was significantly inhibited, but initial transient increase in [Ca2+]i was still remained, which was completely abolished by CPA. In constant Ca2+ levels, SPC induced significant augmentation of the pCa6.3‐induced force but S1P induced little or no augmentation. Pretreatment of fasudil significantly inhibited SPC‐induced contraction. SPC induced several folds increase in Thr696 and Thr853 phosphorylation of MYPT1 but S1P did not significantly affect. Our results suggest that SPC‐induced contraction shows gradual and sustained pattern but S1P‐induced contraction shows initial transient and secondary sustained pattern. Furthermore, activation of RhoA/ROCK pathway and phosphorylation of MYPT1 may be key mechanisms for SPC‐induced contraction but elevation of [Ca2+]i may be a key mechanism for S1P‐induced contraction.
The CNS‐MAP‐Controller hypothesis: A new mathematical model for long‐term control of mean arterial pressure (MAP)
Authors: Averina Viktoria , Othmer Hans , Fink Gregory , Osborn John W
Published: June 2021

We present a new mathematical model in which the long‐term control of MAP is dominated by the central nervous system (CNS). The circulation is modeled as 2 parallel vascular beds; “renal” and “non‐renal”. The model assumes kidney function which fully adapts to chronic changes in MAP. The output of the “CNS‐MAP Controller” regulates sympathetic nerve activity (SNA) to renal and non‐renal arteriolar resistances and venous unstressed volume. Currently, the excitatory drive of the CNS‐MAP‐Controller consists of a synergistic interaction of circulating mineralocorticoids and plasma sodium. The model does not include primary renal dysfunction or “whole body autoregulation” as proposed in the Guyton‐Coleman model. Also, in contrast to the Guyton‐Coleman model in which one hemodynamic profile is proposed to underlie all forms of hypertension, our model suggests that several hemodynamic profiles associated with increased SNA are possible with either no change or decreased total blood volume. These hemodynamic profiles are consistent with published hemodynamic studies in experimental and human essential hypertension. This model supports the hypothesis that dysregulation in the brain, rather than kidney, can be responsible for the pathogenesis of hypertension. Supported by NIH Grant HL064178
Polymer Intravascular Sensors for In Vivo Dynamic Shear Stress Analysis
Authors: Ai Lisong , Yu Hongyu , Rouhanizadeh Mahsa , Qiu Xiaotun , Patel Darhsin , Kloner Robert , Kim Eun Sok , Hsiai Tzung
Published: June 2021

Fluid shear stress modulates the focal nature of atherosclerosis. We tested whether a biocompatible sensor would resolve temporal variations in shear stress in a New Zealand White (NZW) rabbit model. Titanium (Ti) and Platinum (Pt) were deposited on the silicon wafer and patterned to form the sensing elements that were embedded in a polymer known as parylene C. The sensor was deployed into the aorta of NZW rabbits (n=5) via the catheter by the femoral cut‐down. Based on the heat transfer principle, the temperature perturbation altered the resistance of the sensing elements, from which shear stress was calibrated. Fluoroscope was used to visualize the position of sensor and contrast dye was injected to assess the catheter position in relation to the arterial wall. In a focal region of the abdominal aorta above the left renal artery, the time‐averaged shear stress was 15.33 dyn/cm2, and that of the Computational fluid dynamic (CFD) code based on the properties of Non‐Newtonian fluid CFD code was 13.42 dyn/cm2. Both direct measurement and CFD code followed the similar trends within experimental errors. We demonstrate the feasibility of polymer sensors to acquire shear stress with spatial and temporal variations in vivo. Improvement in controlling the orientation and position of the sensors would allow for investigation of hemodynamics in the aortic arch.
L‐Citrulline Inhibits Chronic Hypoxia‐Induced Pulmonary Hypertension in Newborn Piglets
Authors: Fike Candice , Ananthakrishnan Madhumita , Kaplowitz Mark , Barr Frederick , Summar Marshall
Published: June 2021

Decreased Nitric Oxide (NO) production may contribute to the development of chronic hypoxia‐induced PHT. NO is produced from arginine by nitric oxide synthase. Citrulline is metabolized to arginine and may increase the amount of arginine available for NO production. Our goal was to determine if supplementation with L‐citrulline inhibits chronic hypoxia‐induced PHT in newborn pigs. Two day old pigs were kept in hypoxia (H, n=15) or normoxia (controls, C, n=5) for 10 days. Some (n=5) H pigs received oral L‐citrulline (0.13 mg/kg/dose bid). Catheters were placed to measure pulmonary artery pressure (Ppa), left ventricular end diastolic pressure, cardiac output by thermodilution and exhaled NO concentration (ppb). Ppa (cm H2O) and pulmonary vascular resistance (PVR, cmH2O.ml−1.min−1.kg−1) were higher in untreated H (Ppa 39±3, PVR 0.13±0.01) than C (Ppa 21±3, PVR 0.04±0.01) piglets. Ppa and calculated PVR in citrulline‐treated H (Ppa 27±1, PVR 0.07±0.004) did not differ from C. Values for exhaled NO were lower in untreated H (1.5 ±0.2) than C (3.4±0.6) but did not differ between citrulline‐treated H (2.5±0.4) and C pigs. Thus, L‐citrulline increases NO production and inhibits the development of PHT in piglets exposed to 10 days chronic hypoxia. We speculate that enteral supplementation with L‐citrulline may limit pulmonary vascular changes in neonates who have prolonged periods of hypoxia. Supported: AHA‐SE Affil
Superoxide mediates central cardiovascular effects of Urotensin II in spontaneously hypertensive rats
Authors: Lu Ning , Zhou Li , Yao Tai , Zhu Yi‐Chun
Published: June 2021

Recent evidences indicate that central Urotensin II may participate in the regulation of cardiovascular functions however, their central mechanism remained unknown. we tested the hypothesis that reactive oxygen oxidant species (ROS) mediate central cardiocvascular effects of U‐II, Tempol (a superoxide dismutase mimetic) pretreatment would prevent these effects of U‐II. Experiments were completed in urethane‐chloralose‐anesthetized, Wistar‐Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). UII receptors expression in the rostral ventrolateral medulla (RVLM) and Nucleus Tractus Solitarius (NTS) in brain were determined by immunochemistry. Brain superoxide levels were tested in U‐II treated rats using dihydroethidium (DHE) staining. Mean arterial pressure (MAP), Heart Rate (HR) recorded after intracerebroventricular (icv) infusion of U‐II or Tempol. The following observations were made: 1) Expressions of U‐II receptors of RVLM and NTS were increased in SHR rats compared with WKY rats. 2) Brain superoxide levels were enhanced in U‐II‐treated rats compared with aCSF‐treated rats. 3) icv administration of U‐II (10 nmol/10 ul) significantly increased MAP and HR, Tempol pretreatments eliminated these effects of U‐II. These results suggest that superoxide mediates central neural actions of U‐II and provide evidence for a novel relationship between U‐II and ROS.