bluuebunny/crossref_metadata_2025 huggingface dataset:
Trace amine dependent vasoconstriction in porcine coronary artery
Authors: Herbert Amy Angharad , Broadley Kenneth , Kidd Emma , Ford William
Published: June 2021

Trace amines tyramine and β‐phenylethylamine (β‐PEA) contract porcine coronary artery. The mechanism involved in the vascular effect of trace amines have not been fully established. We hypothesized that vasoconstriction by tyramine and β‐PEA are mediated by recently discovered trace amine associated receptors (TAARs). The responses to tyramine and β‐PEA were investigated in vitro in porcine isolated left anterior descending coronary artery rings. Tyramine and β‐PEA induced concentration‐dependent vasoconstrictions. The maximum response and EC50 value for p‐tyramine were unaffected by pre‐treatment with antagonists for adrenoceptors, histamine, dopamine or seratonin receptors. A substantial response to β‐PEA also remained in the presence of these antagonists. The EC50 value for β‐PEA was unaffected by any of the antagonists tested. Antagonists for TAARs are currently unavailable. Biogenic amine receptors thought to be activated directly or indirectly by trace amines, were therefore eliminated one by one until it remained that only a trace amine associated receptor (or a similar receptor) could be mediating the vasoconstrictor response observed to the trace amines tested. In conclusion, these results show that tyramine and β‐PEA cause vasoconstriction in porcine coronary artery that is independent of biogenic amine receptors and could be mediated by TAARs. Supported by the British Heart Foundation.
Increased activation of BKCa channels prevents endothelial dysfunction in coronary arteries from ouabain‐induced hypertensive rats
Authors: Briones Ana M , Cobeño Laura , Padilha Alessandra S , Cogolludo Angel , Vassallo Dalton V , Perez‐Vizcaino Francisco , Salaices Mercedes
Published: June 2021

Chronic ouabain administration induces hypertension. The aim of this study was to compare the endothelium‐dependent relaxation in coronary arteries (CA) isolated from ouabain‐treated versus non‐treated Wistar rats. In 5HT precontracted arteries, acetylcholine (Ach) induced similar relaxant responses in CA from both groups, and this relaxant effect was abolished by the nitric oxide synthase inhibitor L‐NAME. However, when arteries were contracted with high KCl or preincubated with the BKCa channel inhibitor iberiotoxin, but not with the Kv channel blocker 4‐aminopyridine, the relaxation elicited by Ach was more reduced in ouabain‐treated than control rats. Similar results were obtained with the NO donor DEA‐NO in endothelium‐denuded CA. In CA myocytes isolated from ouabain‐treated, but not from non‐treated rats, addition of DEA‐NO (1 μM) markedly increased the amplitude of the iberiotoxin‐sensitive current in the whole range of test potentials. This effect was prevented by the soluble guanylate cyclase inhibitor ODQ. Our results suggest that an increased activation of BKCa channels by NO prevents endothelial dysfunction in CA from ouabain‐treated rats. Supported by AGL2004‐06685/ALI, SAF2005‐03770, Fundacion Mutua Madrileña, SAF2006‐02376 and RECAVA RD06/0014/0011
Effects of Thromboxane Synthase Inhibition on the Contribution of L‐Type Ca2+ channels to Hypoxia‐induced Neonatal Pulmonary Hypertension
Authors: Hirenallur‐S Dinesh K. , Haworth Steven T. , Leming Jeaninne , Madden Jane A. , Rusch Nancy J. , Gordon John B.
Published: June 2021

Pulmonary hypertension (PH) in neonatal piglets caused by chronic hypoxia (CH, 10% O2) is associated with increased thromboxane (TxA2) production, an upregulation of L‐type Ca2+ (CaL) channels, and elevated Ca2+‐dependent tone. Thus, we hypothesized that TxA2 synthase inhibitors may attenuate the development of CH‐induced PH by limiting the upregulation of vascular CaL channels. Two day old piglets were exposed to 3 wks of normoxia (N), CH, or CH treated by the TxA2 synthase inhibitor, furegrelate (CH‐F; 3 mg/kg tid). Subsequently, in‐vivo and isolated lung hemodynamics and CaL channel expression was evaluated. In‐vivo pulmonary vascular resistance index (PVRI) averaged 0.035±0.002 (N), 0.108±0.01 (CH) and 0.061±0.007 (CH‐F). Thus, furegrelate partially normalized PVRI in CH piglets. Furegrelate also normalized pulmonary arterial pressure (PAP) in isolated lungs of CH piglets (N, 11.8±1.1; CH, 24.5±3.1; CH‐F, 14.3±0.8 mm Hg). Nifedipine‐sensitive tone accounted for 55% of the 12.7 mm Hg increase in PAP of CH piglets, and furegrelate abolished this anomalous vascular tone. This finding correlated with a 44% reduction in the expression of the pore‐forming α1C subunit of the CaL channel in small pulmonary arteries. Our findings suggest that TxA2 ‐synthase inhibitors ameliorate the development of hypoxia‐induced neonatal PH, possibly by blunting the upregulation of CaL channels. Supported by NIH HL83013‐03
Expression and localization of adenylyl cyclase isoforms in primary mouse airway smooth muscle cells
Authors: Xu Cong Feng , Ostrom Rennolds S
Published: June 2021

In asthma, airway smooth muscle (ASM) becomes hyper‐contractile, causing a restriction of airflow. β‐adrenergic receptor (βAR) agonists activate adenylyl cyclase (AC) to increase cAMP production and relaxation of ASM. However, βAR appear to only couple to certain AC isoforms due to compartmentation in lipid rafts and caveolae. We examined the expression and localization of AC isoforms in primary mouse ASM. Enzymatic digestion of airway was followed by selection of single cells using a microscope and micropipette. We then prepared total RNA and analyzed AC expression via RT‐PCR. Of the 9 AC isoforms, we found expression of AC6 and AC3, with lower levels of AC4. AC2 expression was only occasionally detected. We then examined the localization of both native and overexpressed ACÕs by confocal microscopy. After double staining with caveolin‐1 antibody, we observed relatively high co‐localization between AC3 (21.3%) or AC6 (20.2%) and caveolin‐1. The degree of overlap was increased in cells overexpressing either AC isoform via incubation with recombinant adenoviruses (31.4% for AC3 and 29.7% for AC6). By contrast, low levels of co‐localization were observed between AC2 and caveolin‐1 (1.4%), with only a slight increase (9.6%) after infection with AC2 adenovirus. Our data indicate that mouse ASM express AC2, AC3, AC4 and AC6 but that only AC3 and AC6 are significantly co‐localized with caveolin‐1. Supported by HL079166.
The Aquaporin 5 gene polymorphism AQP5‐1364 A/C has no impact on loss of renal function in children following renal transplantation
Authors: Büscher Rainer , Adamzik Michael , Frey Ulrich , Nagel Dominik , Hoyer Peter F.
Published: June 2021

Objectives:

Aquaporins, a family of transmembrane water channels, are expressed in the kidney and are involved in chronic renal allograft dysfunction. A novel gain of function promotor polymorphism in the Aquaporin 5 gene (AQP5‐1364 A/C) has recently been demonstrated to alter AQP5 expression and might therefore contribute to water and salt homeostasis. We investigated whether the AQP5‐1364 A/C SNP is associated with loss of glomerular filtration rate (GFR, ml/min/1.73m2) and blood pressure in children following renal transplantation (RTx).

Methods and Results:

91 children (60 male, 31 female, mean age at RTx 9.6 years, mean follow up time 5.4 years) were included. The slope of GFR was determined by linear regression analysis. Allelic frequencies were not significantly different from healthy controls. However, homozygous C‐allele carriers were not detected in our study group. Loss of GFR and blood pressure were not different between the groups (AQP5‐1364 A/C: slope AA −0.02±0.17 vs. AC −0.22±0.17, p=0.454.

Conclusion:

The AQP5‐1364 A/C promotor polymorphism is not associated with the loss of renal function and blood pressure in children following RTx.
Efficacy of Diuretic Agents in Sprague Dawley Rats
Authors: Obih Patience Ogechi
Published: June 2021

Diuretics are agents that increase urine flow and many are employed in the treatment of different types of cardiovascular diseases such as hypertension. They are useful in mobilizing edematous fluid associated with heart failure, cirrhosis, and kidney disease. Their importance in therapy makes this study critical. The aim of this study is to evaluate the potencies of certain diuretics that act at different sites of the nephron in Sprague Dawley rats. Groups of five rats weighing 180–200 gm were injected individually with each diuretic or diuretic combination, furosemide (8 mg/kg), hydrochlorothiazide (40 mg/kg), acetazolamide (10 mg/kg), aminophylline (30 mg/kg), and furosemide/probenecid (40 mg/kg) by intraperitoneal route and placed in metabolic cages. At intervals of 30 minutes and for 90 minutes running, urine sample from each rat was collected and the volume measured. Saline was used as the control. Furosemide caused the highest amount of diuresis (11.5 ml) followed by a combination of furosemide and probenecid. Hydrochlorothiazide was next after probenecid/furosemide combination. Diuresis with furosemide started within 30 minutes of injection proving to have the fastest onset of action. Furosemide/probenecid combination produced diuresis beyond two hours. Furosemide and furosemide/probenecid proved to be more efficacious than other drugs used in this study, hydrochlorothiazide was next.
DIFFERENTIAL EXPRESSION OF PRO‐APOPTOTIC AND ANTI‐APOPTOTIC GENES DURING DICLOFENAC‐INDUCED OXIDATIVE STRESS‐MEDIATED NEPHROTOXICITY IN VIVO
Authors: Bhatt Shruti , Patel Chirag , Ray Sidhartha D.
Published: June 2021

Diclofenac (DCLF), a prominent prescription NSAID, is widely used for the management of arthritis. In addition, DCLF is also a powerful inducer of oxidative stress and genomic fragmentation, and has limitations on its use due to its nephrotoxic potential. This study investigated whether DCLF‐mediated oxidative stress modulates the expression of pro‐ and anti‐apoptotic genes in mouse kidneys. Female ICR mice (25–35 g), fed ad libitum, were administered a dose of DCLF 200 mg/Kg, po and sacrificed 0, 6, 12, 18 and 24 hr later. Study parameters included serum chemistry (BUN, Creatinine, CK, and ALT), measurement of oxidative stress (MDA, SOD, NO2/NO3) and western‐blot analysis of bcl‐2, bcl‐XL, bad, bax, and p53. DCLF‐induced considerable nephrotoxicity (BUN in mg/dl: Con‐26.35±3 & DCLF‐24hr 90±14; creatinine in mg/dl Con: 0.9±0.1 & DCLF‐24hr 1.5±0.0) and parallel changes in lipid peroxidation, nitric oxide production and SOD activity. The most dramatic changes were: substantial increases in the expression of pro apoptotic bax and bad proteins (at 12, 18, 24hrs), reduction in the expression of anti‐apoptotic bcl‐2 & bcl‐xl genes (at 6, 12, 18, 24hrs), and downregulation of p53. Overall, biochemical changes mirrored the genetic changes. This study established the possible involvement of cell death regulating genes during DCLF‐induced nephrotoxicity; however their clinical significance needs to be determined.
Chronic Exposure to Beta‐Blockers Attenuates Inflammation and Mucous Metaplasia in a Murine Asthma Model
Authors: nguyen long phan , Omoluabi Ozozoma , Parra Sergio , Frieske Joanna , Clement Cecilia , Ho Samuel , Knoll Brian J , Tuvim Michael J , Dickey Burton F , Bond Richard A
Published: June 2021

Based upon analogies to recent changes in the treatment of heart failure, we previously showed that chronic administration of certain ‘beta‐blockers’ decreased airway hyperresponsiveness (AHR) in a murine model of allergic asthma. To elucidate the mechanism for the beneficial effects, we used the same murine model to examine the effects of chronic administration of beta‐blockers on inflammation and mucous metaplasia, cardinal features of asthma that may contribute to AHR. Chronic administration of beta‐blockers reduced the total cell counts and eosinophils in bronchoalveolar lavage (BAL) of antigen‐challenged and sensitized mice. There was also reduced mucin production and goblet cell formation (mucous metaplasia) as determined by periodic acid Schiff's staining. A similar effect was observed by a second laboratory using a different strain of mice (C57BL/6J instead of BALB/cJ), with ~ 90% reduction in eosinophils and mucous metaplasia. These results were confirmed by a decrease in Muc5AC staining of airway epithelium using immunohistochemistry and Muc5AC transcript levels. BAL cytokine levels of IL‐13, IL‐10, IL‐5, and TGF‐â1 were also decreased. These observations suggest that asthmatics may benefit from treatment with beta‐blockers given their ability to reduce various indices of airway inflammation including mucous metaplasia, and inflammatory cells and cytokines in BAL.
Genetic polymorphisms in the RNA polymerase II core promoter and enhancer elements of the UGT1A1 promoter influence activation of its gene transcription
Authors: Li Ye , Buckley David , Wang Shuang , Hart Steven N. , Nakamoto Kaori , Klaassen Curtis D. , Zhong Xiaobo
Published: June 2021

RNA polymerase II core promoter and enhancer/repressor elements are involved in regulation of gene transcription. The objective of this study is to examine whether genetic polymorphisms in these elements can change regulation of gene transcription. Genetic polymorphisms in the UGT1A1 promoter were evaluated, which include polymorphism of (TA)6/7TAA (*1/*28) in the TATA box, and two SNPs (−3279 T>G *60 and .C3156 G>A*93) in the distal enhancer elements. These polymorphisms in 100 human liver samples were genotyped by sequencing or microarray chips. mRNA levels of UGT1A1 and 9 transcriptional factors (PXR, CAR, PPARa, GR, Nrf2, AhR, HNF1a, HFN4a, and CEBPa) were quantified by the bDNA technique. Associations were established by multiple linear regressions with adjustment for confounders. Correlations of gene transcription between UGT1A1 and each of the 9 transcriptional factors were observed in the samples carrying genotypes of *1/*1 and *1/*28. However, such correlations did not exist in the samples carrying combinational genotypes of *28/*28 with *60/*60 or *93/*93, which also associated with significantly lower UGT1A1 mRNA levels. Polymorphisms in both RNA polymerase II core promoter and enhancer elements of the UGT1A1 promoter may have a synergetic effect on regulation of UGT1A1 transcription, but the mechanisms need to be investigated. This work was supported by NIH COBRE 5P20 RR021940.
Lipoteichoic acid modulates hepatic drug metabolizing enzymes by activating the Toll‐like receptor 2 signaling pathway
Authors: Ghose Romi , Guo Tao
Published: June 2021

Background:

Altered expression of drug metabolizing enzymes (DMEs) in inflammation can affect the metabolism and toxicity of drugs. It is known that inflammatory responses are induced in immune cells by Lipopolysaccahride (LPS) and Lipoteichoic acid (LTA), derived from gram −ve and gram +ve bacteria, respectively. We reported that LPS modulates gene expression of hepatic DMEs and their regulatory nuclear receptors (NRs: PXR, CAR and RXRα) by activating Toll‐like receptor (TLR) 4 signaling pathway. We hypothesize that LTA can modulate NR and DME gene expression by activating the TLR2 signaling pathway. Methods: Adult male C57BL/6 mice were i.p. injected with 6 μg/g bw LTA or saline. Livers were harvested and RNA and proteins were analyzed by real‐time PCR and western blotting, respectively.

Results:

LTA treatment for 2h resulted in ∼80% suppression of CAR and target DME genes, Cyp2b10, Cyp2a4 and Sultn. The NR, PXR, and the main phase 1 DME, Cyp3a11 were reduced ∼70% by 4–8 h of LTA treatment. Nuclear protein levels of RXRα were reduced in LTA‐treated mice. LTA injection induced cytokine production and cell‐signaling components in the liver. The effects of LTA on NRs, DMEs, cytokines and cell‐signaling pathways were blocked in TLR2−/− mice, indicating that the effects of LTA on hepatic genes were mediated by TLR2.

Conclusions:

TLR2 is involved in regulating DME gene expression during induction of inflammation by LTA.
Oxidized high‐density lipoprotein inhibits platelet activation and aggregation via scavenger receptor BI
Authors: Podrez Eugene A. , Ashraf Mohammad Z. , Kar Niladri , Ma Yi , Byzova Tatiana V. , Febbraio Maria , Valiyaveettil Manojkumar
Published: June 2021

Oxidatively modified LDL (OxLDL) and HDL (OxHDL) are present in the intima of atheromatous plaques and in plasma. While prothrombotic activity of OxLDL mediated by platelet CD36 is established, the role of oxHDL in thrombosis is not known. We now report that OxHDL, but not native HDL, is a potent inhibitor of platelet activation and aggregation induced by physiological agonists. OxHDL inhibited key platelet activation events including the activation of integrin alpha IIb beta 3, alpha‐granule secretion and dense‐granule release. Oxidized lipoproteins are ligands for scavenger receptors CD36 and SR‐BI, both of which are expressed on platelets. Studies using CD36−/− or SR‐BI−/− platelets and blocking antibodies demonstrated that the anti‐thrombotic activity of OxHDL is mediated exclusively by platelet SR‐BI. Isolated SR‐BI−/− platelets were more responsive to agonists than WT platelets supporting a physiological role for platelet SR‐BI. Platelets from eNOS−/−, Akt‐1−/− and Akt‐2−/− mice were as sensitive to oxHDL as WT platelets, indicating that the OxHDL effect is mediated by a pathway different from the eNOS/Akt pathway. These novel findings demonstrate that HDL, upon oxidation, acquires an anti‐thrombotic activity mediated by platelet SR‐BI and suggest that oxHDL may counterbalance pro‐thrombotic activity of oxLDL. Support was by NIH grants HL077213 (EAP), HL071625 (TVB), and HL70083 (MF).
D‐4F restores proinflammatory HDL profiles back to anti‐inflammatory in hypercholesterolemic LDLr−/− mice
Authors: Ou Jingsong , Xu Hao , Ou Zhijun , Coleman Papa‐Ekow , Ying Rong , Huang Dade , Oldham Keith T
Published: June 2021

D‐4F, an apolipoprotein A‐I (apoA‐I) mimetic peptide, has been shown to improve high density lipoprotien (HDL) function. A recent report indicated HDL proteomics were different between healthy and hypercholesterolemia (HC) patients. To test if D‐4F changes the HDL proteomics, low density lipoprotein receptor‐null (LDLr−/−) mice were fed western diet ±D‐4F for 12 weeks. ApoA‐I was immunoprecipitated from mice plasma and the protein composition analyzed by 2‐D gel electrophoresis and nano‐LC/MS. We observed the 2‐D gel patterns were different among the different groups. The 2‐D gel patterns between control (C) and HC + D‐4F were similar, which were both different from HC alone. More than 200 proteins in each group were identified by nano‐LC/MS. More proteins were common between C and HC + D‐4F than between C or HC + D‐4F and HC. The concentration of atherogenic proteins such as apolipoprotein E and apolipoprotein C IV, was higher in HC than C or HC + D‐4F. More complement proteins (C4, C1q, factor B) were observed in HC than in C and HC + D‐4F. Western blot analysis confirmed proteomics findings. Our data indicate HC shifts HDL proteomics to a profile that is proatherogenic and D‐4F shift the profile to an anti‐atherogenic profile. These provide new insight into how HC impairs HDL function by allowing atherogenic proteins to bind to the HDL particle and how D‐4F improves HDL function by inhibiting binding of atherogenic proteins.
Connexin37 Regulation of Vascular Remodeling Following Ischemic Injury
Authors: Fang Jennifer Shea‐Ying , Simon Alexander M. , Burt Janis M.
Published: June 2021

Gap junction proteins Connexin37 (Cx37) and Connexin40 (Cx40) are strongly expressed in normal vascular endothelium, although their specific function(s) in this tissue are poorly understood. Previously, we have shown by dual‐dye injection of oppositely‐charged dyes that both Cx37 and Cx40 channels are similarly cation‐selective. Despite similarities in permselectivity, expression of Cx37 – but not Cx40 – suppresses proliferation of otherwise connexin‐deficient rat insulinoma cells, suggesting that Cx37 may be uniquely suited to regulating endothelial cell proliferation in vivo. Indeed, animals lacking both Cx37 and Cx40 experience profound vascular abnormalities resembling hemangiomas. Using the unilateral hindlimb ischemia model to induce a vascular remodeling response in wild‐type and Cx37‐deficient animals, we show that revascularization following induction of ischemia (as determined by comparison of vascular density in the ischemic versus non‐ischemic hindlimb) is enhanced (~50%) in Cx37‐deficient animals compared to wild‐type controls. These findings are consistent with the hypothesis that in mature endothelium, Cx37 suppresses cell proliferation and controls remodeling of the vasculature. Further, we hypothesize that the inhibitory effect of Cx37 is temporarily relieved by downregulation of its expression during the revascularization response induced by ischemia. Support: AHA 550158Z (JMB), HL064232 (AMS) and AHA 0715532Z (JSF).
Towards a systematic design of novel endogenous peptide‐based antiangiogenic approaches
Authors: Karagiannis Emmanouil , Popel Aleksander S.
Published: June 2021

To date the quest for the experimental identification of angiogenesis inhibitors has been an empirical time‐consuming process. Selected proteins were proteolytically processed by proteases and the cleaved fragments were tested for their antiangiogenic potency. In this study we introduced a systematic computational and experimental methodology that enabled us to identify and classify novel putative endogenous inhibitors of angiogenesis. The novels peptides are derived from various protein families including thrombospondins, and CXC chemokines, type IV collagen fibrils, as well as coagulation factors, and various kringle‐containing proteins. Testing their activity in vitro in suppressing the proliferation and migration of endothelial cells provided proof of principle for the validity of the computational method. By performing receptor neutralization studies we identified receptors that the novel peptides bind. Using the receptor binding information we designed peptide combinatorial therapeutic strategies. In some cases the combinatorial targeting of specific receptors identified strong synergism. Based on the results from the in vitro screening, we tested the ability of the peptides to suppress angiogenesis in in vivo assays including the subcutaneous basement‐membrane‐extract filled angioreactors assay, the corneal micro‐pocket angiogenesis assay and tumor xenograft models.
Continuous infusion of hyaluronidase reduces systemic glycocalyx volume and associates with increased molecular clearance
Authors: van den Berg Bernard , Meuwese Marijn Clara‐Maud , Stroes Erik Sjoerd Gerard , Vink Hans
Published: June 2021

The endothelial glycocalyx shields the vascular wall from flowing blood and contributes to the permeability barrier.

We hypothesized that continuous infusion of testicular hyaluronidase reduces the glycocalyx, resulting in increased clearance rates for large dextran molecules.

C57Bl6 mice received 10, 25 or 100 U/hr hyaluronidase (intravenously, osmotic minipump) for 3, 7 or 14 days. Systemic glycocalyx volume (Vg) was estimated by tracer dilution using labeled red blood cells and 40kDa dextran. Clearance rate of 40kDa and 500kDa dextran were evaluated.

Three days of hyaluronidase (100 U/hr) first increased Vg to 34.8±11.2 (SD) ml/kg, base‐line Vg 20.1±2.8 ml/kg. Continuation reduced Vg to 8.7±4.3 ml/kg (day 7) and 7.4±14.1 ml/kg at day 14 and was associated with increased clearance rates (min−1), from 0.023±0.002 and 0.0022±0.002 (day 0: 40‐ and 500kDa) to 0.044±0.016 and 0.020±0.005 (day 14: 40‐ and 500kDa). A dose dependent reduction in Vg was observed (day 14: 10.3±1.5 at 25 U/hr, 11.9±5.3 at 10 U/hr).

We conclude that prolonged infusion of hyaluronidase eventually reduces systemic glycocalyx which in turn, results in a reduced permeability barrier.

Supported by NHF 2005T037 to H. Vink and 2006B08 to E. Stroes
Adenosine monophosphate kinase (AMPK) expression and activity in the lung
Authors: Creighton Judy , Insel Paul
Published: June 2021

AMPK activity has been implicated in endothelial cell death and vascular smooth muscle proliferation in hypoxia, suggesting that this metabolic sensor participates in vascular pathology. However, molecular mechanisms of AMPK signaling in the lung are poorly understood. Initial studies revealed that the 2 isoforms of AMPK (AMPKα1 and AMPKα2) are expressed at low levels in the rat lung but that hypoxia induces AMPK expression in an isoform‐ and location‐dependent manner. AMPKα1 expression is predominantly induced in the lung periphery and is associated with capillaries while AMPKα2 is expressed in larger vessels. Selective expression of AMPK isoforms extends to distinct pulmonary cell types: pulmonary artery endothelial cells (PAECs) express AMPKα2 while pulmonary microvascular endothelial cells (PMVECs) express AMPKα1. Smooth muscle cells (SMCs) and columnar epithelia express both isoforms. Studies using PAEC, PMVEC, and SMC cultures revealed that AMPK inhibition increases SMC growth and attenuates PMVEC growth. Immunochemical staining indicated that AMPK α1 co‐localizes with the cell adhesion molecule N‐cadherin near regions of cell‐cell contact in PMVECs, results confirmed in co‐immunoprecipitation assays. In conclusion, AMPK expression and function in the lung exhibits both isoform and cell‐type specificity. Supported by research and training grants from NIH.
Fibroblast‐specific expression of adenylyl cyclase 6 reduces myofibroblast differentiation and protects against bleomycin‐induced pulmonary fibrosis
Authors: Liu Xiaoqiu , Li Fengying , Ostrom Rennolds S
Published: June 2021

Pulmonary fibroblasts (PF) regulate extracellular matrix production and degradation and are critical in maintenance of lung structure, function and repair. cAMP‐elevating agents inhibit cytokine‐ and growth factor‐stimulated myofibroblast differentiation and collagen synthesis in PF and overexpression of adenylyl cyclase 6 (AC6) enhances these responses. Thus, we generated transgenic mice that overexpress AC6 under a fibroblast‐specific promoter, FTS1. FTS1‐AC6+/− mice or littermate controls were given intratracheal instillation of saline or bleomycin (5 mg/Kg) and their lungs were removed after 14 days for pathological analysis and assay of collagen content. H&E and trichrome staining revealed normal lung histology in both FTS1‐AC6+/− and WT mice receiving saline. The WT mice receiving bleomycin showed extensive peri‐bronchial and interstitial fibrosis and collagen deposition. By contrast, FTS1‐AC6+/− mice displayed more limited fibrotic development and lower total collagen content. Blinded pathological scoring of the degree of fibrosis (using a 0–8 scale with 8 being the most fibrotic) indicates a statistically significant effect of the FTS1‐AC6 transgene (WT: 6.4 ± 0.2, FTS1‐AC6+/−: 3.5 ± 0.4, p < 0.001). We conclude that AC6 overexpression inhibits fibrogenesis in the lung by reducing PF‐mediated collagen synthesis and myofibroblast differentiation. Supported by NIH grant HL071781.
Inhibition of Cx43‐induced gap junctional communication on growth of cultured endothelial monolayers
Authors: Houser Sandra Dean , Quadri Sadiqa K. , Bhattacharya Jahar
Published: June 2021

Deletion of the gap junctional protein, connexin 43 (Cx43) blocks endothelial (EC) proliferation and vasculogenesis. However, since Cx43 supports intercellular gap junctional communication (GJC), the growth inhibition is potentially attributable to lack of Cx43 expression, or blockade of GJC. To distinguish between these possibilities, in rat lung microvascular EC (RLMEC) we expressed full length GFP‐Cx43 cDNA containing a mutation on threonine 154 (Cx43T154). In HeLa cells, this mutant blocks Cx43‐dependent GJC, but not Cx43 expression (JBC, 2006. 281:7994‐8009). Both wild type (WT; n=3) and Cx43T154‐expressing (n=3) RLMEC monolayers grew to confluence, indicating that the mutant did not inhibit EC proliferation. To assay GJC, we loaded RLMEC with the intracellular cytosolic fluorescent marker, calcein‐AM. Then in single cells viewed by confocal microscopy, we bleached fluorescence to 50% of initial fluorescence (Io), then determined the fluorescence recovery after photobleaching (FRAP). In WT monolayers, FRAP was 22 ± 6% of Io in 3 min (mean ± SD; n=3). In Cx43T154‐expressing monolayers, FRAP was markedly less at 4.2 ± 3 % of Io (n=3; P<0.05). Hence, expression of the mutant Cx43 markedly impaired GJC. We conclude that inhibition of GJC does not inhibit EC proliferation to form stable monolayers (HL36024, HL57556, HL64896).
MKP‐1 inhibits high NaCl‐induced phosphorylation of p38, but does not inhibit high NaCl‐induced activation of TonEBP: opposite roles of p38alpha and p38delta in activation of TonEBP.
Authors: Zhou Xiaoming , Ferraris Joan D. , Dmitrieva Natasha , Liu Yusen , Burg Maurice B
Published: June 2021

High NaCl acutely activates p38 MAP kinase by phosphorylating it, the phosphorylation presumably being regulated by a balance of kinases and phosphatases. The kinases are known, but identity of the phosphatases is uncertain. Our initial purpose was to identify the phosphatases. We find that in HEK293 cells transient over expression of MKP‐1, a dual specificity phosphatase, inhibits high NaCl‐induced phosphorylation of p38, and that over expression of a dominant negative mutant of MKP‐1 does the opposite. High NaCl regulates MKP‐1 activity through reactive oxygen species, which inhibit MKP‐1 and by reduction of MKP‐1 binding to p38. Since inhibition of p38alpha (p38a) is known to reduce hypertonicity‐induced activation of the osmoprotective transcription factor, TonEBP, we anticipated that MKP‐1 expression might also. However, over expression of MKP‐1 has no significant effect on this activity. The paradox is explained by opposing effects of p38a and p38delta (p38d), both of which are activated by high NaCl. Thus, we find that over expression of p38a increases high NaCl‐induced TonEBP activity, but over expression of p38d has the opposite effect. Also, siRNA‐mediated knock‐down of p38d enhances high NaCl‐induced activation of TonEBP. We conclude that high NaCl inhibits MKP‐1, and that this contributes to activation of p38. However, opposing actions of p38a and p38d result in no net effect on TonEBP activity. Thus, 1) contrary to previous interpretations, activation of p38 isoforms by hypertonicity may not contribute to activation of TonEBP because of opposing effects of p38a and p38d and 2) effects of inhibitors of p38 depend on which isoform is affected, which can be misleading.
The role of phosphorylations in hypotonic nuclear export of OREBP/TonEBP/NFAT5
Authors: Ko Ben C.B. , Xu S.U. , Wong Catherine C.L. , Tong Edith H.Y. , Yates John R , Chung Stephen S.M.
Published: June 2021

The osmotic response element‐binding protein (OREBP), also known as tonicity enhancer‐binding protein (TonEBP) or NFAT5, is hitherto the only known osmo‐sensitive transcription factor that mediates cellular adaptations to extracellular hypertonic stress. Here we showed that nucleocytoplasmic trafficking of OREBP is regulated by Ser155 and Ser158 dual phosphorylation. Alanine scanning mutagenesis revealed that Ser155 is an essential residues in OREBP nucleocytoplasmic trafficking. Tandem mass spectrometry revealed that Ser155, as well as Ser158 of OREBP are phosphorylated in vivo under hypotonic conditions. In vitro phosphorylation assay confirmed the findings from mass spectrometry and further revealed that alanine substitution of Ser155 abolished in vitro phosphorylation of Ser158, whereas an aspartate substitution potentiate the phosphorylation, suggesting that phosphorylation of the two serine residues proceeds in a hierarchical manner in which phosphorylation of Ser155 is a prerequisite for phosphorylation of Ser158. Consistently, alanine substitution of either serine residue prevents nuclear exclusion of OREBP under hypotonic conditions. Our data shed lights into the mechanism that regulates OREBP nucleocytoplasmic trafficking.
FRET assessment of CFTR molecular assembly
Authors: Berdiev Bakhrom K , Tousson Albert , Oosterveld‐Hut Henderika M.J. , Qadri Yawar J. , Cormet‐Boyaka Estelle , Hong Jeong S. , Fuller Cathy M. , Sorscher Eric J. , Lukacs Gergely L. , Benos Dale J.
Published: June 2021

In spite of extensive studies, uncertainty exists regarding the number of CFTR molecules that come together to form a conducting pore. At present, findings point both to monomeric and multimeric assembly of CFTR protein. We evaluated the CFTR assembly by fluorescence resonance energy transfer imaging. FRET exploits the exquisite sensitivity of fluorescence measurements to detect molecular complexes with near angstrom resolution. We assayed for FRET between (i) N‐terminally tagged CFTRs (ECFP‐CFTR and EYFP‐CFTR); (ii) C‐terminally tagged CFTRs (CFTR‐ECFP and CFTR‐EYFP); and (iii) both N‐terminally and C‐terminally tagged CFTRs (ECFP‐CFTR and CFTR‐EYFP). We found no appreciable increase in CFP fluorescence after selective photobleaching of YFP, indicative of no FRET occurrence. Our findings show that the cytoplasmic tails of CFTR are not in sufficient proximity for the occurrence of FRET, suggestive of monomeric organization of CFTR.

Supported by NIH 2RO1‐DK37206‐15, NIH P50 DK53090‐05, NIH RO1‐DK075302, CFF/CFFT.
Novel Regulation of the Epithelial Na+ Channel by Soluble Adenylyl Cyclase in Kidney Collecting Duct Cells
Authors: Hallows Kenneth R. , Pastor‐Soler Nuria M. , Edinger Robert S. , Oyster Nicholas M. , Wang Huamin , Buck Jochen , Levin Lonny R. , Johnson John P.
Published: June 2021

Alkalosis impairs natriuresis, but the mechanisms involved are unclear. In contrast to transmembrane adenylyl cyclases, soluble adenylyl cyclase (sAC) mediates HCO3−‐induced elevation of cAMP in intracellular microdomains. We thus hypothesized that sAC may regulate the epithelial Na+ channel (ENaC) in the kidney. Confocal images of rat kidney revealed co‐immunolocalization of sAC and α‐ENaC in collecting duct principal cells. Mouse polarized kidney collecting duct (mpkCCDc14) cells, which express sAC by immunoblot, were used as an in vitro model system. The specific sAC inhibitor KH7 (60 μM) inhibited amiloride‐sensitive short‐circuit currents (Isc) by >50% within 30 min. Similar ENaC inhibition was found with a catechol estrogen, an alternative sAC inhibitor. Aldosterone (1 μM) doubled ENaC‐dependent Isc over 4 h, an effect that was abolished by KH7. Conversely, 8‐Br‐cAMP overrode the inhibition of ENaC by KH7. Moreover, KH7 fully blocked forskolin‐induced ENaC apical recycling, suggesting that sAC activity is required for this process. Consistent with this idea, neither sAC over‐expression nor KH7 treatment modulated ENaC currents expressed in Xenopus oocytes, where significant ENaC recycling has been shown not to occur. In summary, these results suggest that sAC is a novel regulator of ENaC. sAC activity is necessary for both aldosterone stimulation of ENaC currents and ENaC recycling in CCD cells.
Aldosterone‐dependent and ‐independent effects on renal potassium excretion
Authors: Picard Nicolas , Loffing‐Cueni Dominique , Mihailova Marija , Makhanova Natalia , Smithies Oliver , Wagner Carsten , Loffing Johannes
Published: June 2021

Aldosterone (aldo) is released from adrenal glands upon potassium (K) loading and hyperkalemia. It stimulates renal K excretion via the ROMK K‐channel and via activation of the apical epithelial sodium channel (ENaC) providing the electrochemical driving force for K secretion. Previous studies using adrenalectomized rodent models showed that both aldo‐dependent and ‐independent effects are important for renal K excretion. Here we characterized a mouse model lacking aldosterone synthase and thus endogenous aldo production. Under basal conditions, KO mice are slightly hyperkalemic but excrete similar quantities of K with urine as WT mice. K loading with food (3% and 5% for 2 days) induced kaliuresis in WT and KO mice. KO mice excreted a 3–4 fold higher volume of diluted urine after 5% K‐loading and were mildly hyperkalemic. Immunohistochemistry revealed that K‐loading (3% K) induced a translocation of ROMK and ENaC from intracellular compartments to the apical membrane of the renal collecting system in WT mice. While the translocation of ENaC was lacking, the translocation of ROMK was even more pronounced in KO mice. Thus, the renal adaptation to K‐loading involves an aldo‐dependent translocation of ENaC and an aldo‐independent translocation of ROMK. This defect is likely compensated by increased apical targeting of ROMK, a higher urinary flow and reduced concentration of urine, which possibly increases flow‐stimulated K secretion and prevents life‐threatening hyperkalemia.
M3R stimulates endocytosis of two variants of SLC4A4 in rat ParC5 cell
Authors: Grichtchenko irina I , Rocafort‐Ward Angelique , Reyland Mary , Quissell David , Perry Clint
Published: June 2021

Salivary ion and fluid secretion is regulated by parasympathetic activity mediated through muscarinic M3R receptors in acinar cells. The SLC4 bicarbonate transporters are the major passages for salivary bicarbonate secretion. We previously reported that in Xenopus oocytes, AT1 signaling regulates NBC1 function and surface expression by endocytosis. Here we study M3R signaling regulation of endogenous and recombinant NBC1 in rat parotid acinar cells (ParC5). We localized NBC1 to the basolateral membrane (BLM) in ParC5 cells. We showed that PMA and Carbachol induce redistribution of the cotransporter from the cell surface to endosomes. Inhibition of NBC1 recycling with the carboxylic ionophore monensin also caused significant accumulation of NBC1 in endosomes and a parallel loss of NBC1 from the cell surface. Co‐localization of internalized endogenous NBC1, NBC1‐A‐YFP or NBC1‐B‐GFP with Texas Red‐conjugated transferrin or Early Endosomal Antigen 1 antibody suggests clathrin‐dependent endocytosis within both contituitive and stimulated endocytosis. We showed that PMA and Carbachol induced PKC‐dependent endocytosis of NBC1 by using GF109203X. Using GÖ6976, we eliminated the involvement of PKCαβγ. We then identified that PKCδ is necessary in PMA and Carbachol stimulated endocytosis by knockdown of the endogenous PKCδ by expressing its kinase‐dead mutant. We have also reported that calmodulin antagonist W13 inhibits constitutive recycling of NBC1‐A in Xenopus oocytes. Here we demonstrated that W13 blocks recycling of endogenous NBC1, NBC1‐A‐YFP and NBC1‐B‐GFP in ParC5 cells. Our data demonstrate that NBC1 is constitutively endocytosed through a calmodulin dependent mechanism and that PMA and M3R signaling induce PKCδ‐dependent endocytosis of NBC1‐A/B.
Mucosal carbonic anhydrases and acid sensors regulate the CO2‐induced hyperemia in rat esophagus
Authors: Akiba Yasutada , Mizumori Misa , Kaunitz Jonathan D
Published: June 2021

Heartburn is usually triggered by esophageal luminal acid. We hypothesized that luminal CO2, rather than H+, permeates through the stratum epithelium, interacting with epithelial and sub‐epithelial carbonic anhydrases (CA) and acid chemosensors, producing afferent neural signals. We superfused rat lower esophageal mucosa with pH 1.0 or pH 6.4 high CO2 solution, inhibiting CA activity with methazolamide or benzolamide, TRPV1 with capsazepine, and ASIC with amiloride. We measured interstitial pH (pHint) with carboxyfluorescein iv and blood flow with laser Doppler flowmetry. Perfusion of a high CO2 solution or pH 1.0 induced hyperemia without pHint change. All inhibitors inhibited CO2‐induced hyperemia and decreased pHint. Immunofluorescence localized CA XIV and all ASICs to the prickle cells, CA XII to the basal cells, TRPV1 to the stratum granulosum, and TRPV1 and ASIC3 to the muscularis mucosa. Luminal CO2 reproduced the mucosal response to acid superfusion, suggesting that CO2 diffuses through the stratum epithelium before interacting with epithelial or submucosal acid sensors. Inhibition of the hyperemic response to CO2 by CA, TRPV1 and ASIC inhibitors implicates CA and acid sensors in the transduction of the luminal acid signal. Transepithelial CO2 permeation may explain how luminal H+ equivalents can be rapidly sensed, producing responses such as hyperemia and heartburn.
Regulation of K‐Cl cotransporters by signaling complex involving WNK3, WNK4, SPAK, and protein phosphatases
Authors: de‐los‐Heros Paola , Rivera Jetzabeth , Ponce‐Coria Jose , Vazquez Norma , Gamba Gerardo
Published: June 2021

The K‐Cl cotransporters (KCC1 to KCC4) encompass a branch of the SLC12 family of electroneutral cation‐coupled chloride cotransporters that promotes chloride efflux from the cells. We have previously shown that WNK3 and WNK4 inhibit the activity of KCCs. Elimination of kinase activity (WNK3‐D294A or WNK4‐D318A) not only prevented the inhibitory effect, but turned them into a powerful activators of all KCCs for WNK3‐DA and KCC2 and KCC3 for WNK4‐DA. In the present study we showed in Xenopus laevis oocytes that activation of all four KCCs by WNK3‐DA in isotonic conditions was prevented by co‐injection with wild type WNK4. Similarly, activation of KCC2 or KCC3 by WNK4‐DA was prevented by the presence of wild type WNK3. Thus, both activation by WNK3‐DA or WNK4‐DA and inhibition by WNK3 or WNK4 use the same pathway. Elimination of the SPAK unique binding site of WNK3 or WNK4 significatively reduced the protein‐protein interaction between WNK3 or WNK4 with SPAK, but had no effect upon their KCCs inhibitory properties during cell swelling. However, elimination of the SPAK binding site completely prevented the activation of KCCs in isotonic conditions by both, WNK3‐DA and WNK4‐DA. Since we know that this activation can be prevented by protein phosphatases inhibitors, we propose that KCCs are regulated by a signaling complex that involves WNKs, SPAK and protein phosphatases.
PTH suppresses sodium chloride cotransporter activity by a Mapk‐dependent process
Authors: Ko Benjamin , Cooke Leslie L , Hoover Robert S
Published: June 2021

Recently, we described that Diacylglycerol (DAG) analogues act through Ras guanyl‐releasing protein1 (RasGRP1) stimulation of ERK1/2 MAPK to decrease function and surface expression of the thiazide‐sensitive sodium chloride cotransporter (NCC). However, upstream hormonal mediators of DAG‐dependent NCC suppression have not been identified. Although PTH activates DAG through G‐protein coupled receptor activation of Phospholipase C, PTH regulation of NCC has not been demonstrated. Utilizing a mouse DCT (mDCT) cell line with native NCC activity, we investigated whether PTH may mediate suppression of NCC function through MAPK activation. Thiazide‐sensitive 22Na+ uptakes were measured by incubating mDCT cells for 15 minutes in a Cl−free medium before exposure to a 22Na+containing medium with or without thiazide. Uptakes were determined after 15 minute incubations with vehicle, PTH (10‐7M) alone, PTH + U0126 (MEK 1/2 MAPK inhibitor), and PTH + bisindolylmaleimide (BIM, PKC inhibitor). PTH decreased NCC activity by 43.6 +/− 4.5% (p<0.05 for PTH vs. vehicle). U0126 blocked the PTH‐induced suppression while BIM had no effect on the suppression. This is consistent with our previous data that RasGRP1 and not PKC is the mediator of DAG effects on NCC activity. These studies indicate that PTH decreases NCC function via downstream activation of the MAPK pathway. This work was supported by NIH Grant K08 DK070668.
Endothelin‐1‐induced NOS3 activation is mediated by the PI3K/AKT pathway in the thick ascending limb
Authors: Herrera Marcela , Garvin Jeffrey L.
Published: June 2021

Endothelin‐1 (ET‐1) inhibits NaCl transport by the thick ascending limb (THAL) via increased NO. NOS3 is the primary source of NO in this segment. Phosphorylation of NOS3 at serine 1177 (pNOS3 Ser1177) positively regulates its activity. We hypothesized that ET‐1 stimulates pNOS3 Ser1177 via activation of the PI3K/AKT pathway. We measured: 1) pNOS3 Ser1177 by Western blot; 2) AKT activity using a FRET‐based AKT reporter; and 3) pAKT Ser473. Rat THALs were treated with ET‐1 (1nM) for 15 min. ET‐1 increased pNOS3 Ser1177 by 45 ± 9 % vs. control (p = 0.001; n = 6). Wortmannin (wort., 150nM), a PI3K inhibitor, blocked this effect (Δ + 3 ± 14 % vs. wort. alone, n = 5). The AKT reporter was expressed in the THAL by in vivo gene transfer. The probe is a fusion protein of the AKT consensus phosphorylation site, CFP and YFP (Kunkel et al, JBC. 280(7):5581–7). An increase in the CFP/YFP ratio indicates increased AKT activity. THALs were isolated and FRET measured by fluorescence microscopy. ET‐1 increased the CFP/YFP ratio by 0.16 ± 0.02 fluorescence units (f.u) (p < 0.01; n = 5). Wort. blocked this effect (Δ + 0.01 ± 0.01 f.u; n = 5). ET‐1 increased pAKT Ser473 by 47 ± 14% vs. control (p < 0.001; n = 5). Wort. blocked this effect (Δ + 2 ± 6 % vs. wort. alone). We concluded that ET‐1 acutely phosphorylates NOS3 at Ser1177 via PI3K/AKT activation in the THAL. This could be an important regulatory mechanism of THAL NaCl reabsorption by ET‐1.

HL028982, HL070985
Residue at position 93 of the Na+/iodide symporter (NIS) plays a critical role in Na+/substrate coupled transport
Authors: Maestas Matthew J. , Dohán Orsolya , Paroder Monika , Nicola Juan Pablo , Eskandari Sepehr , Carrasco Nancy
Published: June 2021

The Na+/I− symporter (NIS) mediates Na+‐dependent active I− uptake in the thyroid. We report the electrophysiological characterization of G93R NIS, a clinically‐identified NIS mutation that causes congenital I− transport defect, and other substitutions at position 93. Wild‐type (WT) NIS and G93N, G93T, and G93R mutants were expressed in X. laevis oocytes and the two‐electrode voltage clamp technique was used to study steady‐state and presteady‐state kinetic properties of the transporters. Similar to WT NIS, the G93N and G93T mutants exhibited electrogenic Na+‐dependent I− transport; however, the mutants displayed reduced apparent affinity for I−(>500 μM vs. 30 μM). In contrast to WT NIS, G93N and G93T NIS mutants exhibited electrogenic transport of perchlorate, an environmental pollutant and well‐known NIS inhibitor. Steady‐state kinetics revealed the apparent affinity of the mutants for perrhenate and perchlorate to be 20–30 μM. Steady‐state Na+ kinetics showed a sigmoidal dose‐response relationship with all substrates examined (I−, perchlorate, perrhenate, and chlorate). The Na+ apparent affinity (~30 mM) and the Na+ Hill coefficient (~2) of the mutants were similar to those of the WT. Presteady‐state currents of the mutants were similar to those of the WT. Although trafficked to the cell surface, G93R NIS was inactive. The data suggest that residue 93 may play a key role in coordinating substrate and/or Na+ in the binding pocket. NIH grants S06 GM53933 to S.E. and DK41544 and CA098390 to N.C.
Ablation of Uncoupling Protein‐3 Does Not Inhibit Palmitate Export from Mouse Heart Mitochondria
Authors: Matlib Mohammed Abdul , Gerber Lamar K
Published: June 2021

Uncoupling protein‐3 (UCP3) has been implicated in the export of long‐chain free fatty acid (FA) from mitochondria. However, there is no direct evidence of FA export from mitochondria. If UCP3 is involved in FA export from mitochondria then its ablation should inhibit the process. Employing a novel technique developed in our laboratory that directly measures FA export from isolated mitochondria, we sought to determine whether or not the rate of export of palmitate generated in the matrix from palmitoyl‐carnitine is inhibited in mitochondria isolated from UCP3 knockout (UCP3KO) mouse hearts. UCP3KO mouse heart mitochondria had no detectable UCP3 protein. However, the UCP2 level was similar to wild type mouse heart mitochondria. The mitochondrial thioesterase‐1 (MTE‐1) which generates FA in the matrix was increased by ~30% in UCP3 knockout compared to WT mice. The rate of generation of palmitate from palmitoyl‐carnitine in mitochondria was also increased by ~25%, but there was no inhibition in the rate of export of palmitate from mitochondria of UCP3KO mouse hearts. We conclude that UCP3 does not exclusively export FA from mitochondria.