bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of hsp27 Phosphorylation Increases Interaction with Hic5 in Vascular Myocytes
Authors: Martin Jody Lee , Bossuyt Julie , Fontaine Jean‐Marc , Welsh Michael J , Bers Don , Donofrio Anthony
Published: June 2021

The small heat shock protein, hsp27, is a cytosolic ubiquitously expressed, stress responsive chaperone. It is phosphorylated rapidly in response to a variety of stressors via the p38 MAP kinase pathway. Hsp27 has been associated with and modulates a variety of essential proteins such as actin and cytochrome c. It has also been shown to bind to and be modulated by the paxillin homologue, Hic‐5 (hydrogen peroxide inducible clone 5, also known as ARA55 for androgen receptor activator 55kda). We sought to examine the nature of this interaction via co‐expressing hsp27‐cfp and Hic‐5‐mcitrine fusion proteins and confocal FRET methodology. Both fusions were adenovirally mounted to allow myocyte transduction. Of note, in neonatal cardiomyocytes and smooth muscle cells (SMCs) there was no measureable interaction under basal conditions. With 60 minute pretreatment of SMCs with the specific p38 inhibitor, SB203580 at 10uM, there was a marked increase in F485 vs basal and vs pretreatment with the MEK inhibitor PD90859 at 30 uM (63±3.9%*vs −5±5% vs −6.5±2.6%, respectively, n=9, *p=0.001 by ANOVA, post‐hoc Bonferroni). In contrast, inhibitor pretreatment produced no significant FRET in cardiomyocytes nor did the use of mutagenized nonphosphorylatable 3Ahsp27‐cfp in SMCs. We conclude that only nonphosphorylated hsp27 species interact with Hic‐5 and in specific cell types.
Pulmonary venous hypertension causes differential expression of developmental genes in the lung
Authors: Hoffmann Julia , Kuebler Wolfgang M
Published: June 2021

Pulmonary venous hypertension (PVH) is a frequent consequence of left sided heart disease and causes extensive lung vascular remodeling which leads to right ventricular failure. Functional genomics underlying this structural remodeling in PVH are unknown but present potential targets for therapeutic strategies. Developmental genes have been implicated in the pathophysiology of lung cancer and asthma but their role in vascular remodeling and pulmonary hypertension is unclear.

PVH was induced in rats by supracoronary aortic banding 8 weeks prior to investigations. For genomic analyses, RNA was extracted from isolated lungs of control and PVH rats and whole rat genome microarrays were performed.

Out of 31100 arrayed genes, 231 were significantly up‐ or downregulated more than 3‐fold in PHV. These comprised genes involved in vascular regulation and inflammation such as ETB receptor, heme oxygenase 1 and interleukin‐1 alpha but also developmental genes like frizzled related protein, Jun D and semaphorin 6A. Differential expression of selected genes was verified by RT‐PCR and confirmed by Western Blot and 2D‐PAGE.

PVH causes differential expression of genes involved in vascular regulation and development. Functional effects of this genomic regulation may contribute to the pathology of PVH and suggest a potential role for developmental genes in vascular remodeling.

Sponsored by EU‐IP “Pulmotension”
Endothelin receptor (ETR) blockade combined with phosphodiesterase‐5 (PDE5) inhibition prevents right ventricular hypoxia in Pulmonary Hypertension
Authors: Mouchaers Koen Thijs Bert , Wong Yeun‐Ying , Schalij Ingrid , Jaspers Richard T , van Nieuw Amerongen Geerten P , Vonk‐Noordegraaf Anton , van der Laarse Willem J
Published: June 2021

Background

Pulmonary hypertension (PH) may cause right ventricular (RV) hypoxia, which may contribute to RV failure.

Goal

To study the effects of current PH therapies on the presence of hypoxia in the RV.

Methods

PH was induced in Wistar rats by monocrotaline (MCT) (40 mg/kg). From day 14 to 28, rats were treated with Bosentan (ETR antagonist, 300 mg/kg/day), Sildenafil (PDE5 inhibitor, 10 mg/kg/day) or both (all n=3). Healthy rats served as controls (n=3). 1.5 hour prior to sacrifice hypoxyprobe (pimonidazole, 60 mg/kg) was administered i.v. Cryosections (5 μm) of the hearts were incubated with hypoxyprobe antibody and for succinate dehydrogenase (SDH) activity. RV capillary density and cardiomyocyte cross‐sectional area (CSA) were determined.

Results

PH significantly increased RV pimonidazole binding, and cardiomyocyte CSA, and decreased capillary density, and SDH activity (all p<0.05 vs control). Single treatment with Bosentan or Sildenafil had no effect, whereas combination treatment normalized all parameters (p<0.05 vs MCT rats). Pimonidazole binding related linearly to CSA (r=0.6), SDH activity (r=−0.7), and capillary density (r=−0.8) (all p<0.05).

Conclusion

Hypoxia in the RV of PH rats is caused by cardiomyocyte hypertrophy, a lowering in mitochondrial activity and a diminished capillary density, and can be prevented by combining ETR blockade and PDE5 inhibition.
Interaction of nitric oxide and reactive oxygen species in the development of hypoxic pulmonary hypertension
Authors: Chovanec Milan , Hnilickova Olga , Smolkova Pavlina , Herget Jan
Published: June 2021

Chronic hypoxia damages peripheral pulmonary vessels and causes hypoxic pulmonary hypertension (HPH). According to our hypothesis, the release of reactive oxygen species (ROS), nitric oxide (NO) and products of their interaction contribute to the pathogenesis of HPH. Experimenting with chronic hypoxic adult male rats, we tested the effect of NO and ROS interaction. Hypoxic rats were treated solely with SOD mimetic Tempol (group T, n=6) and NO donor Molsidomine (group M, n=9) or in combination (group MT, n=6) during first 7 days of exposure to 3 weeks hypoxia (FiO2=0.1). Seven untreated hypoxic rats (group H) and seven normoxic controls (group C) were used.

Pulmonary artery blood pressure (PAP), and the weight of RV, LV+S were measured. PAP was significantly decreased in group M (P<0.05) and group T (P<0.05) but not in group MT compared with group H. In all hypoxic groups the right‐to‐left heart weight ratio was significantly increased compared to group C, and groups receiving Tempol (T, MT) had significantly (P<0.01 and P<0.01 respectively) less increase in weight of RV or whole heart compared with group H.

Molsidomine or Tempol added alone (but not in combination) at the beginning of exposure to chronic hypoxia reduces HPH. Tempol alone or in a combination reduces of hypoxia‐induced right heart hypertrophy.

Supported by Cardiovascular Research Center MSMT 1M 0510, Grants: GACR 305/05/0672 and GAUK 7780/2007
Attenuated PLC‐Dependent Activation of Voltage‐Gated Ca2+ Entry in Pulmonary Endothelium Following Chronic Hypoxia
Authors: Paffett Michael L. , Resta Thomas C. , Kanagy Nancy L. , Walker Benjimen R.
Published: June 2021

Chronic hypoxia (CH)‐induced pulmonary hypertension is associated with reduced endothelial cell (EC) receptor‐operated Ca2+ (ROC) entry. Voltage‐gated Ca2+ channels (VGCC) are present in pulmonary ECs and are activated by protein kinase C (PKC). Although VGCCs may participate in ROC entry, neither the role of phospolipase C (PLC) nor the effect of CH on this entry pathway are known. We hypothesized that CH reduces phospholipase C (PLC)‐induced Ca2+ influx through VGCCs in pulmonary ECs. Thus, we assessed both KCl and ATP‐induced Ca2+ entry in freshly isolated, fura‐2 loaded ECs obtained from control and CH rats (4 wk at 0.5 atm). Consistent with earlier findings, KCl‐induced Ca2+ entry was diminished following CH. Furthermore, KCl‐mediated Ca2+ influx in control ECs was attenuated by the T‐type VGCC inhibitor, mibrefradil (MBF; 10 μM), but not by the L‐type inhibitor diltiazem (50 μM). Similarly, ATP‐induced Ca2+ entry in control ECs was reduced to the level of CH ECs by MBF and was partially inhibited by the ROC blocker SKF96365 (20 μM). Neither of these antagonists affected Ca2+ entry in CH ECs. Finally, the PLC inhibitor U73122 (1 μM) abolished ATP‐induced Ca2+ influx in ECs from both control and CH rats, but the inactive analog U73343 was without effect. These data suggest that purinergic receptor stimulation in pulmonary ECs activates T‐type VGCCs through a PLC‐dependent mechanism that is diminished following CH.
Experimental Hypertension is Associated with Differential Expression of Angiotensin‐(1–12) in Heart of Hypertensive and Normotensive Rats
Authors: Jessup Jewell A , Habibi Javad , Trask Aaron J. , Chappell Mark C. , Nagata Sayaka , Kato Johji , Kitamura Kazuo , Sowers James R. , Ferrario Carlos M.
Published: June 2021

Low expression of angiotensinogen in the heart has been construed as indicating a circulating uptake mechanism to explain the local effects of angiotensin II in the tissue. The recent identification of angiotensin‐12 [Ang‐(1–12)] and our localization of the propeptide in cardiac myocytes led us to hypothesize that Ang‐(1–12) may be an alternate substrate for local angiotensin production. Immunolocalization of Ang‐(1–12) in the heart of SHR and WKY rats [11 weeks, n = 8], revealed robust and mottled Ang‐(1–12) staining found predominantly in ventricular myocytes with less, but distinct staining found in the medial layer of intra‐coronary arteries and vascular endothelium. Quantitative analysis of the stained sections, captured using laser confocal microscopy, demonstrated that the average cardiac grayscale staining intensity of the fluorescent signal was 3‐fold higher (P < 0.0001) in SHR compared to WKY paralleling tissue content measurements of Ang‐(1–12) which demonstrated a 47% higher (P < 0.05) content of the propeptide in the heart of SHR compared to WKY. These new findings suggest that Ang‐(1–12) functions as a preprohormone for tissue‐specific renin‐angiotensin systems. Furthermore, the differential manifestation between the normo‐ and hypertensive models is indicative of a role for Ang‐(1–12) in the evolution of a genetic form of hypertension. Funding provided by NIH (HL‐51952).
Temporal analysis of signaling pathways associated with the development of renovascular hypertension
Authors: Grande Joseph Peter , Cheng Jingfei , Yin Ping , Zhou Wei , Warner Gina M. , Gray Catherine E.
Published: June 2021

The objective of this study was to identify signaling pathways leading to the development of interstitial fibrosis and tubular atrophy in the stenotic kidney and compensatory hypertrophy/hyperplasia of the contralateral kidney in a murine 2 kidney 1 clip model of hypertensive nephrosclerosis. Mice were studied 2, 5, and 11 weeks after undergoing surgery to establish unilateral renal artery stenosis (RAS). RAS animals, but not sham controls, became hypertensive within 1 week. Compensatory enlargement of the contralateral kidney was associated with a transient increase in proliferation (as assessed by PCNA and Mib‐1), p‐ERK, caspase 3, p27, p53, and TGF‐β expression at early time points, with return to baseline levels by 11 weeks. Tubular atrophy in the stenotic kidney was associated with a persistent increase in p‐ERK, Mib‐1, p53, p21, and p27 expression through 11 weeks. Proliferative activity was observed in tubular epithelial cells and in inflammatory cells. The atrophic kidneys had little apoptotic activity (caspase 3, TUNEL staining). The atrophic kidneys showed progressive increases in TGF‐β and α‐SMA expression and extracellular matrix deposition throughout 11 weeks. Interstitial inflammation was associated with increased MCP‐1 expression. Mechanisms underlying this differential response to unilateral RAS await definition.
Importance of the chemokine RANTES in the development of angiotensin II‐induced hypertension and vascular dysfunction
Authors: Budzyn Klaudia , Lob Heinrich E , McCann Louise A , Goronzy Jorg J , Weyand Cornelia M , Harrison David G , Guzik Tomasz J
Published: June 2021

Recent studies in our laboratory have revealed a major role for the T cell in the development of angiotensin II‐induced hypertension and associated vascular dysfunction, in parallel with increased levels of the chemokine, RANTES, in blood vessels. These observations suggest an important role for RANTES in stimulating T cell migration during the development of angiotensin II‐induced hypertension. Hence, we have sought to determine the role of RANTES in the development of hypertension and vascular dysfunction induced by angiotensin II. C57Bl/6 and RANTES−/− mice were infused with angiotensin II (490 ng/min/kg) for 14 days, with the hypertensive response to angiotensin II being markedly blunted in RANTES−/− relative to C57Bl/6 mice (P<0.05). Moreover, whilst angiotensin II infusion caused severe impairment of endothelium‐dependent relaxation in isolated aortic segments from C57Bl/6 mice, it had no such effect on relaxation responses of segments from RANTES−/− mice. This preserved endothelial function occurred in parallel with lower superoxide production in these vessels relative to those from C57Bl/6 mice (P<0.05), as well as reduced T cell infiltration. These observations demonstrate that RANTES contributes significantly to the hypertensive response and vascular dysfunction induced by angiotensin II. Thus, selective suppression of RANTES might represent a novel strategy for the treatment of the vascular complications associated with the development of hypertension.
Venous compliance and wall distensibility in the venous compartments of the lower limb in men
Authors: Lindenberger Marcus , Zachrisson Helene , Neider Daniel , Länne Toste
Published: June 2021

Objectives: Lower limb venous compliance is of hemodynamic importance for orthostatic challenge. The qualities of different venous compartments is however unknown. The aim was to study venous buffering compliance (vBC) as well as venous wall distensibility (vWD) in deep (femoral vein, FV) and superficial large veins (great saphenous vein, GSV), as well as to assess calf venous compliance (vC).

Methods: Strain‐gauge plethysmography was used to assess vC. Diameter (D), vBC and vWD of the FV and GSV were examined with ultrasound in 15 young (YM, 25±1 yr) and 14 old (OM, 70±2 yr) males. D was measured at rest and after venous stasis (6 min, 60 mmHg), and vein area (A) calculated. Pressure‐Area curves (absolute, mm2, and relative, %) during a linear pressure decrease were produced, and vC, vBC and vWD calculated using a quadratic regression equation.

Results: vC was greater in YM, P < 0.05. vBC was greater in GSV in YM than OM, P < 0.01. D was larger in FV than GSV in YM and OM, P < 0.0001, and vBC was greater in FV than in GSV irrespective of age, P < 0.01. However, when correcting for venous size (vWD), greater vWD was found in GSV than in FV in YM as well as OM, P < 0.05.

Conclusions: Calf venous compliance was greater in young as compared to old men. Femoral vein had higher venous buffering compliance than great saphenous vein, due to its greater size. When correcting for size, great saphenous vein showed greater wall distensibility than femoral vein.
Vascular endothelial function is selectively, positively related to leg/hip fatness in healthy postmenopausal women
Authors: Pierce Gary L. , Eskurza Iratxe , Anderson John E. , Seals Douglas R.
Published: June 2021

Vascular endothelial dysfunction develops with age and contributes to increased risk of cardiovascular disease (CVD). Abdominal body fat increases, but peripheral body fat decreases, CVD risk particularly in postmenopausal women (PMW). We hypothesized that peripheral body fat is associated with enhanced endothelium‐dependent dilation (EDD) in PMW. In young men and women (n=62, age 24±5y; 45M/17F), EDD (brachial artery flow‐mediated dilation) was not related to total or peripheral body fat (DXA) or to waist:hip ratio (WHR), a measure of abdominal fat. In middle‐aged and older healthy men (n=51, age 62±1y), EDD was inversely related to WHR ratio (r=‐0.29, P=0.04), but not to total or peripheral body fat. In contrast, in healthy PMW (n=27, age 60±1y) EDD was positively related to total fat (r=0.44, P<0.05), hip circumference (r=0.49, P=0.01) and leg/hip fat (r=0.47, P=0.01), but not to arm fat or WHR. EDD was greater in subgroups (n=7/group) of PMW with higher vs. lower leg/hip fat (7.1±3 vs. 4.1±2 %, P<0.01) independent of serum risk factors, but was not significantly (P>0.05) different in subgroups of PMW differing in total body fat or WHR. Plasma estrogen concentrations were higher in a subgroup of PMW with greater vs. lower leg/hip fat (P=0.03). Among healthy PMW, leg/hip body fat is consistently associated with enhanced EDD, independent of differences in conventional CVD risk factors. NIH AG006537, AG013038, AG022241
Matrix‐targeted green fluorescent protein for quantitative monitoring of mitochondrial morphology in living cells
Authors: Nomura Yasutomo , Takayama Toshifumi , Sato Michihiko , Takahashi Eiji , Feng Zhonggang , Nakamura Takao
Published: June 2021

Mitochondrial morphology within a cell appears to be correlated with cellular function. Quantitative measurement of its morphology is therefore essential in order to understand the coupling in detail. In addition to mitochondria‐selective dye such as rhodamine 123 (RH123), a recent progress in genetic engineering allowed the microscopic observation of mitochondria with a matrix‐targeted green fluorescent protein (GFP). Here we evaluated these labeling procedures for the ability to analyze mitochondrial morphology quantitatively. In contrast to no cytosolic GFP, fluorescence of RH123 in cytosol was intense and peripheral mitochondria with weak fluorescence were masked by thresholding without any filters. Although applying a top hat filter improved binarized images for RH123 remarkably, the mitochondrial structures differed slightly from those in raw images. When form factor, aspect ratio, mean area and number of mitochondrion per cell were analyzed from the binarized images, these parameters for GFP were stable to 16 times of repetitive recordings but mean area for RH123 decreased by 30% probably due to photo‐bleaching. We concluded that expression of matrix‐targeted GFP was the optimal procedure to monitor mitochondrial morphology quantitatively because of photo‐stability and precise localization.
Serine protease‐induced decrease in epithelial ion permeability is dependent on an intact actin cytoskeleton
Authors: Swystun Veronica , MacNaughton Wallace K.
Published: June 2021

The serine proteases trypsin, chymotrypsin and elastase induce a sustained increase in transepithelial electrical resistance (RTE) when applied to the apical surface of polarised epithelial cell monolayers that is reflected by decreases in Na+ and Cl− paracellular permeabilities. Paracellular permeability is controlled at the intercellular tight junctions and the adherens junctions, with the epithelial actin cytoskeleton linked to proteins comprising both. We show that manipulation of the actin cytoskeleton with latruculin B and jasplakinolide reduces the protease‐induced increase in RTE by 92 ± 5 and 50 ± 8% respectively. Immunocytostaining for F‐ and G‐ actin indicate a shift in the filamentous actin pool. Inhibition of Rho kinases decreased baseline RTE but did not inhibit the protease‐induced increase in RTE.
Regulation of Na+ transport in H441 cells by AMPK and PIP2
Authors: Mace Oliver John , Baines Deborah L
Published: June 2021

We have shown that the AMP mimetic AICAR activates the metabolic sensor AMP‐activated protein kinase (AMPK) and inhibits Na+ transport in H441 human lung cells. As PIP2 is reported to be required for ENaC activation, we examined whether these pathways converge to regulate Na+ channel activity in H441 cells. Monolayers were mounted in Ussing chambers in physiological salt solution. AICAR (2 mM) rapidly reduced amiloride‐sensitive short‐circuit currents (ISC‐AMIL) by 49.0 ± 7.6 % (P < 0.05, n = 8). Compound C, an inhibitor of AMPK, rescued ISC‐AMIL. Purinergic stimulation by UTP (100 μM) inhibited ISC‐AMIL by 49.2 ± 7.5 % (P < 0.05, n = 8) via depletion of membrane PIP2 as recovery was abolished by blocking PIP2 resynthesis with wortmannin (10 μM). Neomycin (5 mM) which sequesters PIP2 inhibited ISC‐AMIL by 59.7 ± 12.6 % (P < 0.05, n = 4) and there was no further inhibition with UTP. Following treatment with either UTP or neomycin, AICAR further reduced ISC‐AMIL to 72.9 ± 4.0 % and 77.1 ± 7.1 % (P < 0.05, n = 4), respectively. U‐73122 (10 μM), an inhibitor of phospholipase C elevated ISC‐AMIL 1.3 fold (P = 0.13, n = 4) and AICAR inhibited the elevated current by 47.7 ± 5.6 % (P < 0.05, n = 4). These data indicate that AICAR inhibits Na+ transport via mechanism/s additional to membrane depletion of PIP2. This work was supported by the BBSRC.
An Aldosterone‐sensitive Basolateral P2X4 Receptor Stimulates the Renal Epithelial Sodium Channel
Authors: Bao Hui Fang , Yu Ling , Helms My , Eaton Douglas C
Published: June 2021

Our results suggest that one way aldosterone can stimulate ENaC is via elevation of P2X4 receptor expression and trafficking to the basolateral membrane of A6 cells. We found that application of either ATP or 2‐methylthio‐ATP (2‐meSATP, a selective P2X receptor agonist) to the basolateral bath significantly elevated ENaC activity in cell‐attached patches formed on the apical membrane of aldosterone‐conditioned A6 cells. In contrast, no effect was observed when 2‐meSATP was applied to the apical bath, or when aldosterone was deprived from the culture medium for 72 h. Furthermore, a P2X4‐like channel was recorded in cell‐attached patches formed on the basolateral membrane of the flipped A6 cell monolayer. The effect of basolateral 2‐meSATP on ENaC activity was blocked by either chelation of extracellular Ca2+or inhibition of PI 3‐kinase. Western blot experiments showed that deprivation of aldosterone for 72 h strongly reduced the P2X4 receptor expression. Furthermore, confocal microscopy data showed that the P2X4 receptor is likely to be located in the basolateral membrane, which is confirmed by basolateral surface biotinylation of ENaC.

This work was supported by NIH grants HL071621‐01 and DK37963‐16 awarded to DCE.
Regulation of ENaC in the rat collecting duct by endothelin‐1
Authors: Pochynyuk Oleh , Bugaj Vladislav , Stockand James D
Published: June 2021

The epithelial sodium channel (ENaC) plays a critical role in setting systemic salt and water balance and thus, blood pressure. Activity of this channel is limiting for Na+ reabsorption at the renal collecting duct. Cell signaling in response to endothelin (ET‐1) via the endothelin B receptor decreases ENaC activity in heterologous expression systems. Moreover, as shown by the Kohan laboratory, targeted deletion of ET‐1 and the endothelin B receptor in mouse collecting duct causes hypertension associated with sodium retention. This hypertension is exacerbated by a high sodium diet implicating dysfunctional ENaC regulation. Thus, we test here the hypothesis that ET‐1 modulates ENaC activity in the isolated, split‐open rat collecting duct. In addition, we define the cellular signaling pathway underpinning such regulation in native collecting duct. We find that ET‐1 rapidly decreases ENaCopen probability in the rat collecting duct. As in model cells, pretreatment with a selective Src family kinase inhibitor (PP2) but not its inactive analogue (PP3), blocked ET‐1 action on ENaC. Moreover, inhibition of the endothelin receptor blocked ET‐1 action. Following ET‐1 treatment, ENaC remains in the membrane in a closed/inactive state for increases in apical PI(4,5)P2 levels surmounts endothelin‐dependent inhibition. We conclude that ET‐1 through endothelin B coupled to Src tyrosine kinases decreased ENaC activity in native collecting duct cells by rapidly decreasing channel open probability. These results demonstrate that ET‐1 signaling through Src via the endothelin receptor is a major negative regulator of ENaC activity in native renal epithelialcells.
Dynamin regulates UT‐A1 urea transporter membrane expression
Authors: Huang Haidong , Zhuang Jieqiu , Fröhlich Otto , Yang Yuan , Klein Janet D , Cai Hui , Sands Jeff M , Chen Guangping
Published: June 2021

Dynamin plays a direct role in endocytosis, synaptic vesicle recycling, and caveolae internalization. The urea transporter UT‐A1, located in the kidney inner medullary collecting duct (IMCD), is important for urine concentrating ability. In this study, we found that both UT‐A1 and dynamin are present in caveolae isolated from HEK293 cells by OptiPrep gradient ultracentrifugation. The direct relationship of UT‐A1 and dynamin was identified by co‐immunoprecipitation. Dynamin was precipitated by UT‐A1 antibody when both UT‐A1 and dynamin were transfected into 293 cells. However, phosphorylation does not enhance the binding of UT‐A1 and dynamin. UT‐A1 has both cytosolic amino and carboxyl termini and a large intracellular loop. We therefore tested of which of the three domains dynamin bound. The three intracellular fragments of UT‐A1 were prepared and labeled by 35S methionine by in vitro translation with rabbit reticulocyte lysate. Pull down study showed dynamin specifically binds to the intracellular loop of UT‐A1, but not the N‐ and C‐termini. Cell biotinylation experiments of UT‐A1 demonstrated that co‐expression of dynamin and UT‐A1 in 293 cells resulted in a decrease of UT‐A1 cell surface expression. Conversely, cells expressing dynamin mutants deficient in GTP binding (K44A) revealed a significant increase of UT‐A1 protein cell surface accumulation. Our data suggest that a dynamin‐dependent pathway might be an important mechanism for the regulation of urea transport activity in vivo.
Nuclear Proteomics of the Inner Medullary Collecting Duct (IMCD)
Authors: Tchapyjnikov Dmitry , Li Yuedan , Pisitkun Trairak , Knepper Mark A.
Published: June 2021

Mechanisms of vasopressin‐induced transcriptional regulation in the IMCD are poorly understood. The low abundance of many transcriptional regulators has been a limiting factor in their identification in proteomic studies. To address this, we developed an approach for the purification and enrichment of IMCD nuclear proteins based on differential centrifugation and detergent extraction. Immunoblotting demonstrated a forty‐fold enrichment of the nuclear marker BRG1, while the cytosolic marker aldose reductase was de‐enriched fifty‐fold and the membrane marker AQP2 was de‐enriched ten‐fold. We used 1‐D SDS‐PAGE LC‐MS/MS to identify IMCD nuclear proteins. Two types of protein identification algorithms were employed (SEQUEST and InsPecT), both coupled to a target‐decoy methodology to limit false‐positive identifications to less than 2%. We also designed a suite of JAVA‐based bioinformatics software, called APIEX, for the filtering, analysis, and annotation of these proteomic data sets. With this approach, we identified over 2300 proteins, including many transcription factors with potential relevance to vasopressin signaling (including CREB1, GATA3, STAT1, PAX8, NF‐κB, YY1, KRL7a, Smad 1, and Smad 4). The identified proteins add to the known IMCD proteome (http://dir.nhlbi.nih.gov/papers/lkem/imcd/) and lay the groundwork for a more comprehensive systems biology study of the collecting duct.
AVP causes transient formation of cAMP and activation of phosphodiesterase activity in MDCK cells
Authors: Froehlich Otto , Blount Mitsi A. , Yang Yuan , Klein Janet D. , Sands Jeff M.
Published: June 2021

Exposure of MDCK cells to forskolin (FSK) and vasopressin (AVP) raises intracellular cAMP levels by activating adenylate cyclase (AC). FSK (5 μM) raised, over 10 min, the cellular cAMP approximately ten‐fold to a constant new level. The response to AVP (10−8M) was transient, with cAMP reaching a maximum (5‐fold) after about 5 min and then decaying. The transient cAMP response contrasts with the slow activation (30–40 min) urea flux in MDCK cells. In the presence of the phosphodiesterase (PDE) inhibitor, IBMX, the AVP‐induced cAMP spike was amplified and lasted longer. IBMX also enhanced the AVP‐stimulated urea flux. This enhancement was also achieved with the PDE4 inhibitor rolipram (2–20 μM) but not the PDE1 inhibitor vinpocetine (10–200 μM).

We observed an approximately 2‐fold increase of PDE activity when assaying lysates of AVP‐treated MDCK cells. It appears that protein kinase A is involved in activating PDE, as H89 lengthened the AVP‐induced cAMP transient. Furthermore, AVP and FSK combined caused a very large cAMP transient (10× larger than AVP alone) that returned to levels slightly above those found for FSK alone. Overall, these data suggest a negative feedback mechanism in MDCK cells in which AVP not only activates AC but also, with a delay, PDE to reduce cAMP to a lower steady‐state level. Activation of PDE involves protein kinase A, which is known to be activated by the AVP‐stimulated cAMP
Localization of Facilitated Urea Transporters to Tubular Segments in the Bundle and Sinus Zones of the Kidney of the Euryhaline Stingray, Dasyatis sabina
Authors: Dempsey Adair Marie , Lacy Eric , Janech Michael , Miller Donald , Ploth David , Fitzgibbon Wayne
Published: June 2021

Renal urea reabsorption underpins the osmoregulatory strategy of elasmobranch fishes (sharks, skates and rays). Two urea transporter isoforms (strUT‐1 and strUT‐2) have been identified from the kidney of the Atlantic stingray. strUT‐1 is a carboxy extended form of strUT‐2. We hypothesized that these isoforms play specific roles in urea absorption and are expressed at different tubular sites. To test this hypothesis we used immunohistochemistry to localize strUT expression along the nephron of Atlantic stingrays. Five micron serial paraffin sections were cut from paraformaldehyde fixed kidneys of rays maintained in harbor water (850 mOsmol/kg H2O). Sections were incubated with affinity‐purified antibodies that recognized either the C‐terminal of strUT‐1 or a sequence common to both strUT‐1 and strUT‐2. In the presence of the strUT‐1 antibody, positively stained tubular segments were restricted to the last loop in the bundle zone (the intermediate segments). In contrast, numerous positively stained tubular segments were observed in both bundle and sinus zones of sections incubated with the common antibody. We conclude that 1) the strUT‐2 isoform is widely expressed along the nephron of the Atlantic stingray kidney and is the primary isoform involved in urea reabsorption, and 2) the localization of strUT‐1 to intermediate segments in the bundle zone suggests that it plays a distinct role in urea reabsorption.
Transcriptional Profiling of Native Inner Medullary Collecting Duct Cells from Rat Kidney
Authors: Uawithya Panapat , Pisitkun Trairak , Ruttenberg Brian E. , Knepper Mark A.
Published: June 2021

Vasopressin acts on the renal inner medullary collecting duct (IMCD) to regulate water and urea transport. To obtain a ‘parts list’ of gene products expressed in the IMCD, we carried out mRNA profiling of freshly isolated rat IMCD cells using Affymetrix Rat 230 2.0 microarrays with approximately 31,000 features. 7913 annotated transcripts were found to be expressed above background in the IMCD cells. We have created a “IMCD Transcriptome Database” to make the results publicly accessible. Among the 30 transcripts with the greatest signals on the arrays were three water channels: aquaporin‐2, aquaporin‐3, and aquaporin‐4, all of which have been reported to be targets for regulation by vasopressin. In addition, the transcript with the greatest signal among members of the solute carrier (SLC) family of genes was the UT‐A urea transporter (Slc14a2), which is also regulated by vasopressin. The V2 vasopressin receptor was strongly expressed, but the V1a and V1b receptors did not produce signals above background. Among the 200 protein kinases expressed, the serum‐glucocorticoid regulated kinase (Sgk1) had the greatest signal intensity in the IMCD. Altogether, the results combine with proteomics studies of the IMCD to provide a framework for modeling complex interaction networks responsible for vasopressin action in collecting duct cells. Source of research support: Z01‐HL001285.
Long term vasopressin regulation of renal AQPs and urea transporters in mice with a reduced urinary concentrating ability
Authors: Nelson Sarah , Cai Qi , Keck Maggie , Tse Jonathan , Brooks Heddwen L
Published: June 2021

AQP1 null mice (AQP1 KO) fail to generate a hypertonic medullary interstitium and have a urinary concentrating defect. This study used AQP1 KO mice to investigate how long term vasopressin (AVP) regulates renal aquaporins (AQP) and urea transporters (UTA) in the absence of high osmotic stress. In AQP1 KO mice 6 days of AVP infusion slightly increased urine osmolality (856 ± 33 mOsm vs 752 ± 20 mOsm, P≤0.05). AVP decreased AQP3 mRNA (0.70 ± 0.07 vs 1.00 ± 0.10, P≤0.05) in the inner medulla and did not change AQP3 protein in either the cortex or inner medulla. AVP did not change AQP2 mRNA expression, but increased AQP2 protein (187 ± 12.5 vs 100 ± 9.1%, P≤0.05) in the inner medulla. In the cortex AQP2 mRNA and protein increased (mRNA: 1.68 ± 0.14 vs 1.00 ± 0.07; protein: 143 ± 13.9 vs 100 ± 6.6%, P≤0.05). AVP increased UTA1 mRNA and protein in the inner medulla (mRNA: 2.09 ± 0.1 vs 1.00 ± 0.22; protein: 184 ± 30.0% vs 100 ± 18.1%, P≤0.05). AVP increased collagen 1α1 chain mRNA in the inner medulla and cortex (1.78 ± 0.23 vs 1.00 ± 0.16; 1.66 ± 0.21 vs 1.00 ± 0.24, respectively, P≤0.05) and collagen III mRNA in the cortex (1.87 ± 0.21 vs 1.00 ± 0.23, P≤0.05). The increases in collagen expression did not occur in AVP‐infused wild type mice. We conclude that reduced osmolality in the renal medulla of APQ1 KO mice affects long term AVP‐induced AQP3 and collagens I and III mRNA and AQP 3 protein expression, but does not affect AQP2 and UTA1 protein expression.
Altered conformational activity and cleavage of CD18 integrins on the human neutrophil surface exposed to fluid shear stress
Authors: Shin Hainsworth Yeakun , Simon Scott , Schmid‐Schonbein Geert
Published: June 2021

CD18 integrins are critical regulators of neutrophil adhesion and migration. Previously, we reported that shear stress inhibits neutrophil pseudopod formation while simultaneously reducing membrane expression of CD18 through cleavage by cathepsin B. The role of CD18 cleavage and bond detachment in shear‐induced pseudopod retraction, however, remains uncertain. To address this, we examined control of CD18 surface expression and shifts in affinity during shear‐induced pseudopod retraction. Using antibodies specific for activated CD18 and a FRET reporter for LFA‐1 (a CD18 integrin), we demonstrated that fluid shear induces shifts in the CD18 ectodomain to an open‐extended conformation associated with ligand binding. Western blots and immunofluorescence revealed cleavage of the extracellular domain of CD18 by cathepsin B in the presence, but not absence, of either shear stress or PMA. Furthermore, cathepsin B was necessary for neutrophil pseudopod retraction in response to shear. Together these results suggest a link between CD18 surface levels and shear‐induced pseudopod retraction involving conformational shifts in the CD18 ectodomain that increases its susceptibility to cathepsin B proteolysis. Moreover, these results point to an anti‐inflammatory role for fluid shear due to blood flow to maintain neutrophils in a rounded, non‐adhesive state under physiological conditions.
Role of Lasp‐1 in regulation of cell motility and migration
Authors: Zhang Han , Chen Xunsheng , Chew Catherine S.
Published: June 2021

Lasp‐1 is a phosphorylated signaling protein that is over‐expressed in several cancers. Lasp‐1 is present in focal adhesions (FAs) and binds to the FA protein, zyxin. In cancer cell lines, depletion of Lasp‐1 inhibits cell migration and depletes zyxin from FAs. To define the effects of loss of Lasp‐1 in normal cells, we compared responses of mouse embryonic fibroblasts (MEFs) from Lasp‐1−/− and wild type (WT) littermates. Lasp‐1−/− MEFs migrated faster in wound‐healing assays and attached more rapidly to extracellular matrices. FA numbers were significantly increased in Lasp‐1−/− MEFs, whereas zyxin localization was unchanged. Zyxin expression was also unchanged, suggesting that zyxin redistributes to FA from other cellular compartment(s). Interestingly, zyxin−/− MEFs have also been found to migrate faster than WT MEFs, and Mena/VASP proteins are lost from FAs in these MEFs. We hypothesize that Lasp‐1 and zyxin, and possibly other focal adhesion proteins, interact to regulate normal cell migration. The loss of either Lasp‐1 or zyxin disturbs this balance allowing for increased migration and attachment. The striking contrast in the functional effects of loss of Lasp‐1 in nontransformed cells as compared to cancer cell lines identifies distinct differences in regulatory mechanisms associated with motility and attachment in these cellular models. (Supported by NIH R01 DK31900).
Novel Function of Cysteine‐rich Secretory Protein from Naja Atra Venom:CRISP‐a induces pro‐inflammatory responses of vascular endothelial cells
Authors: Wang Yu‐Ling , Chiu Jeng‐Jiann , Wu Wen‐Guey
Published: June 2021

Cysteine‐rich secretory proteins (CRISPs) have been identified in diverse organisms, including epididymis, salivary glands, and snake venom. There is no report to show physiological function of CRISPs. We investigated the role of CRISP from Naja atra (CRISP‐a) in inflammatory reponses of endothelial cells (ECs). CRISP‐a induced the expression of adhesion molecule intercellular adhesion molecule‐1 (ICAM‐1), vascular adhesion molecule‐1 (VCAM‐1), and E‐selectin, and chemokine growth related oncogene‐α (GRO‐α), in human umbilical vein ECs in a dose‐ and time‐dependent manner. Treatment of ECs with CRISP‐a induced phosphorylations of extracellular signal‐regulated kinase (ERK), c‐Jun‐NH2‐terminal kinase (JNK), and p38, and nuclear factor‐κB (NF‐κB)‐DNA binding activity in ECs. Pre‐treatment of ECs with heparinase, an enzyme that cleaves sulphational sites of heparan sulphate on the EC surface, inhibited the CRISP‐a‐induced GRO‐α expression, suggesting that heparan sulfur on the EC surface plays significant roles in mediating EC responses to CRISP‐a. Our findings elucidate a mechanism by which CRISP from snake venom induces adhesion molecule expression in ECs, thereby playing inflammatory roles in vascular biology.
Biochemical characterization, intracellular localization, and stage‐specific expression of a new type of membrane‐bound β‐glucosidase from Physarum polycephalum
Authors: Hayase Masato , Yubisui Toshitsugu , Minami Yoshiko
Published: June 2021

Physarum polycephalum, one of the true slime molds, has a unique life cycle including the diploid plasmodium. The plasmodia express three forms of β‐glucosidase: an intracellular soluble, membrane‐bound, and extracellular enzyme. Specially, we focus on the membrane‐bound β‐glucosidase, BglM1, of which molecluar mass is 130 kDa. Glycosyl hydrolase familly 3 domain is found within the N‐terminal half of BglM1. Notably, the C‐terminal half of BglM1 contains two calx‐β motifs known as calcium binding sites, an RGD sequence, and a transmembrane region. Thus, Physarum BglM1 is a new type of β‐glucosidase that differs from all previously identified family 3 enzymes. The expression of BglM1 was found only in the plasmodia and microplasmodia during life cycle. The observation by immunofluorescence microscopy indicated that BglM1 is located on the cell membrane. The molecular mass of native BglM1 in microplasmodia was estimated to be 230kDa instead of 130kDa calculated from the amino acid sequence. During BglM1 was purified, its apparent size decreased from 230 to 130kDa gradually. However, when BglM1 was purified in the presence of calcium ion, the molecular mass of enzymes was held on 230kDa. These results suggest that calcium ion influences the conformation of BglM1. Based on these results, we will discuss on the function of BglM1.
SAPHIR: a collaborative multi‐scale, multi‐resolution Core Model Environment for the Physiome—with a prototype core model of blood pressure regulation and fluid homeostasis
Authors: Thomas S. Randall , Hannaert Patrick , Baconnier Pierre , Fontecave Julie , Guillaud François , Le Rolle Virginie , Maziere Pierre , Tahi Fariza , Zehraoui Farida , Abchiche Nadia , Hernandez Alfredo
Published: June 2021

We present the current state of the SAPHIR project, a multi‐resolution core modeling environment (CME) in the spirit of the IUPS Physiome, with application to a prototype core model based on a modular implementation of the classic systems model by Guyton et al. (1972 Ann. Rev. Physiol. 34:13–44) and its extension by Ikeda et al. (1979 Annals Biomed. Engin. 7:135–166). This core model targets short‐ and long‐term regulation of blood pressure and homeostasis of body fluids and major solutes. The aim is to provide a collaborative modeling environment enabling plug‐and‐play construction of integrated systems models with lumped‐parameter sub‐models at the organ/tissue level yet also allowing focus on cell‐ or molecular‐level detailed models embedded in the larger core model. Thus, in silico exploration of gene‐to‐organ‐to‐organism scenarios is possible while keeping computation time manageable. The CME is built on the M2SL toolbox, a multi‐scale, multi‐resolution, multi‐mode open source package developed in C++ by one of us (AH). Associated with the CME is an ontology‐based database allowing exploration of the modules, parameter values, and equations.

In parallel with the CME implementation of the core model, we also present stand‐alone implementations in Berkeley Madonna (Ikeda model) and Simulink (Guyton model).
The effect of denervation on the molecular clock in skeletal muscle
Authors: McCarthy John Joseph , Miyazaki Mitsunori , Wolff Gretchen , Esser Karyn Ann
Published: June 2021

We recently identified the circadian transcriptome in mouse skeletal muscle. The primary objective of this study was to test the hypothesis that innervation is required to maintain oscillation of the circadian molecular clock in muscle. A second objective was to determine if the phase of the molecular clock is the same in different muscles. Using the Period‐2:Luciferase mouse, the tibialas anterior was denervated (d‐TA) by sectioning the common peroneal nerve on one leg with the contralateral leg serving as a sham operated control. Following two weeks of denervation, various muscles and liver were collected from three animals every four hours for 24 hr. RT‐PCR analysis revealed that the expression pattern of components of the molecular clock (Bmal1, Per2 and Clock) as well as Myod1, a clock‐controlled gene, was altered in the d‐TA compared to i‐TA. Luciferase activity, reflecting Per2 protein expression, of the d‐TA was significantly elevated at each time point in comparison to inneravted‐TA (i‐TA), though the time of peak expression remained unchanged. In the heart, diaphragm and liver, peak luciferase activity peaked four hours later than what was observed in hindlimb muscles suggesting the phase of the clock is slightly different in these tissues. Collectively, these results indicate loss of neural input causes alterations in the molecular clock in adult skeletal muscle.

KAE (AR045617) and JJM (AR053641)
Blue light phase advances entrained rhythms of brain temperature and sleep in rhesus monkeys
Authors: Robinson Edward L , Fuller Charles A
Published: June 2021

Blue light sensitive retinal ganglion cells and their neural projections appear to be the main pathway for the effects of light on circadian entrainment and alerting. The effects of blue light on rhythms and sleep in a 24 hour light‐dark cycle were studied in a rhesus monkey model. Six individually housed and unrestrained animals were entrained in cool‐white fluorescent light (WH) for 12 hours daily (LD 12:12), then exposed to 4 days of LD12:12 with blue LED lamps (BL). Both sources averaged 6.1 x 1013 quanta cm−2s−1 at eye level. Performance was measured using the Psychomotor Test System (Georgia State Univ.). Brain temperature (Tbr) and sleep were recorded via biotelemetry. Sleep stages from the last two days of WH and BL were scored in 30‐second epochs. The average fraction of time in sleep over 24‐hours, as well as during L or D, did not differ between WH and BL. Shallow slow‐wave sleep (stages 1 and 2) and deep slow‐wave sleep (stages 3 and 4) also showed no significant differences. The fraction of time in REM sleep over 24‐h was significantly elevated as was REM during D. Acrophases from sine fits showed a statistically significant advance of the phase angle of entrainment in Tbr, in the first two days of BL. Acrophases and average waveforms of sleep stages from the subsequent two days show apparent phase advances and a slight increase in daytime sleep, but did not differ significantly.

(Supported by NASA Grant NNJ04HF44G)
Progressive development of circadian rhythmicity and differential expression of clock genes in neonatal miniature pigs
Authors: Hong Yonggeun , Song Jongwook , Lee Youngjeon , Park Sookyeong
Published: June 2021

Melatonin secretion is stimulated by the dark and inhibited by night. The suprachismatic nuclei (SCN) of the hypothalamus have melatonin receptors and melatonin may have a direct action on SCN to influence “circadian” rhythms. Recent evidence also shows that the circadian system of primate infants is responsive to light at very premature stages and that low‐intensity lighting can regulate the developing clock. After birth of neonatal miniature pigs (mini‐pig), in this study, there is progressive maturation of the circadian system outputs, with pronounced rhythms in sleep‐wake and hormone secretion generally developing after 2 wks of age. Showing the importance of photic regulation of circadian phase in neonatal mini‐pig, exposure of premature neonatal mini‐pig to low‐intensity cycled lighting results in the early establishment of rest‐activity patterns that are in phase with the 24‐hour light‐dark cycle. With the continued elucidation of circadian system development and influences on physiology of mini‐pig and illness, it is anticipated that consideration of circadian biology will become an increasingly important component of neonatal care in animals. [Supported by BioGreen21 Program (Code#20070401034006), Republic of Korea].
Effects of underwater breathing apparatus on ventilatory parameters at rest
Authors: Alain Duvallet , Emilie Duvallet , Michele Beaudry , Annick Bouchez‐Buvry
Published: June 2021

The aim of the study was to determine the effect of breathing through a self contained underwater breathing apparatus (SCUBA) on the ventilatory parameters at rest. SCUBA imposed separated inspiratory and expiratory external resistances and increased the dead space. Volumes and maximal flow‐volume loops were randomly performed with and without SCUBA in 160 men and 125 women aged of 30.9 ± 15.1 years. Data were compared by t paired test, ANOVA and ANCOVA.

SCUBA did not influence maximal vital capacity (4,15 ± 1,02 vs 4,15 ±1,04 l; p = 0,95), increased the expiratory reserve volume (1,24 ± 0,53 vs 2,36 ± 0,78 l; p<0,0001) and decreased inspiratory capacity (2,91 ± 0,83 vs 2,36 ± 0,78 l; p<0,0001). Scuba decreased significantly all parameters of flow‐volume loops. The results were independent of age, sex, and height.

Breathing through a scuba lead to an airway obstruction and shifted the expiratory resting volume in the inspiratory capacity. At high volume, the pulmonary elastic recoil strengths are increased that allowed the expiration easy and inexpensive despite the applied external resistances. Although reduced dynamic lung compliance, the weak external inspiratory resistances and inspired air pressure made the inspiration easy. The available surface area of the valves in the scuba was greater than 50mm2 which was shown to be a threshold to compromise alveolar ventilation (Wasserman, 1995). However, the effect of these modifications on alveolar ventilation and gas exchanges must be study.