bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduction of heat shock protein 27 phosphorylation inhibits the development of intimal hyperplasia
Authors: Komalavilas Padmini , Lopes Luciana B , Flynn Charles R , Smoke Christopher , Seal Brandon , Panitch Alyssa , Brophy Colleen M
Published: June 2021

Development of intimal hyperplasia involves smooth muscle cell proliferation, migration as well as extracellular matrix deposition. These responses require dynamic reorganization of the actin cytoskeleton, a process known to involve HSP27expression and phosphorylation. In this study, we evaluated the ability of a cell permeant peptide inhibitor of the MAPKAP kinase II (MK2i) to decrease the phosphorylation of HSP27 and its effect on the development of intimal hyperplasia in vitro. Human saphenous vein (HSV) rings were placed in 8‐well chamber slides, and maintained in RPMI 1640 medium with 30% FBS for 14 days at 37°C /5% CO2. They were either untreated or treated with MK2i (5 or 10 μM), SB 203580 (25 μM, an inhibitor of the upstream kinase, P38 MAPK), or recombinant HSP27 (3μM). The rings were sectioned, stained, and the intimal thickness was measured. HSP27 phosphorylation was evaluated by Western blot. MK2i and SB reduced the phosphorylation of HSP27 by 39±17% and 58±22% respectively (p < 0.05). MK2i at 10 μM, but not at 5 μM, significantly (p <; 0.05) reduced the intimal thickness by 43±15%; a reduction of 51±10% was observed after treatment with SB. On the other hand, treatment with recombinant HSP27 increased the intimal thickness by 39±27%. These results demonstrate that reduction of HSP27 phosphorylation by MK2i treatment is a potential strategy to inhibit the development of intimal hyperplasia. Support: NIH RO1 70715
Receptor recognition specificity of plasminogen for the novel plasminogen receptor, Plg‐RKT
Authors: Parmer Caitlin M. , Baik Nagyung , Miles Lindsey A.
Published: June 2021

Plasminogen (Plg) receptors promote Plg activation to arm cells with plasmin to promote both thrombolysis and cell migration. Plg‐RKT is a new type II integral membrane Plg receptor on leukocytes and other tissues. Our objective was to define the recognition specificity of Plg for Plg‐RKT. The C‐terminal peptide of Plg‐RKT was coupled to microtiter wells. Biotinylated Plg was incubated with the immobilized peptide and detected with streptavidin‐HRP. Plg bound to the peptide in a concentration‐dependent manner (IC50=5 nM). In specificity controls, Plg binding was competed by unlabeled Plg and no binding to the reverse peptide was detected. Plg binding was inhibited in a dose‐dependent manner by the soluble peptide, but was not affected by a mutant peptide with the C‐terminal K replaced with A. A peptide with the proximal upstream R replaced with K was a more effective competitor than the wild type peptide. Human Plg also recognized the C‐terminal peptides of murine, rat, and bovine Plg‐RKT's that contain the same R6K substitution. Plg domains containing disulfide‐bonded kringles (K) inhibited the interaction with the following order of effectiveness: Plg>K1‐3>K5>K4. In conclusion, the interaction of Plg‐RKT with Plg requires the presence of a C‐terminal lysine and can be modulated by a proximal upstream basic residue. Furthermore, kringle domains within the Plg molecule interact with the C‐terminus of Plg‐RKT.
Tissue Engineering the Aortic Valve Spongiosa Using Matrigel‐Cell‐Scaffold‐Composites (MCSCs)
Authors: Eldred Jordan Rachel , Rogers Kem , Boughner Derek
Published: June 2021

The middle layer of the aortic valve, termed the spongiosa, consists mainly of glycosaminoglycans (GAGs). Its function is to serve as a shock absorber and resist compression forces between the two outer layers of the valve, and is therefore a necessary component of the bioengineered valve. MCSCs were created by seeding radial artery cells (RACs; 2×106/ml) mixed with endothelial growth media and Matrigel onto small intestinal submucosa, and incubated at 37°C for 0, 1, 2 or 3 weeks. Composites were stained with Alcian Blue for GAGs and immunohistochemistry was used to detect the core protein decorin. Thickness of constructs and cell proliferation were quantified, and gelatin zymography was performed to test for extracellular matrix remodeling. Constructs stayed intact for a minimum of three weeks and stained positively for GAGs and decorin. As culture time increased, MCSC thickness increased. Cell density was stable across all culture times. Zymography revealed the presence of both latent and active matrix metalloproteinase 2. In summary, these composites have shown they are capable of producing GAGs, along with one of their associated core proteins, can lay down new matrix, maintain a constant rate of proliferation, and are likely to be engaging in matrix remodeling. These findings support the use of Matrigel and RACs in our bioengineering efforts. This research is funded by The Heart and Stroke Foundation of Ontario.
Nucleus accumbens dopamine and it's role in positively reinforcing aggression
Authors: Couppis Maria Helena , Kennedy Craig H.
Published: June 2021

Rationale: We studied whether mesocorticolimbic dopamine (DA) in the nucleus accumbens (NAc) mediates the rewarding properties of aggression.

Objective: To address this issue, we studied the effect of administrating specific DA receptor antagonists into the NAc on aggression and general movement.

Methods: A residentintruder model of aggression was used with male mice. An operant conditioning paradigm was used to establish a baseline rate of nose pokes to contingently access aggression as a positively reinforcing stimulus. We analyzed whether microinfusions into the NAc of SCH‐23390, a D1 like DA receptor antagonist, or sulpiride, a D2 like DA receptor antagonist, altered aggression and/or movement. Open‐field tests were used to further assess the effects of SCH‐23390 and sulpiride on movement.

Results: Infusing either SCH‐23390 or sulpiride decreased responding for contingent aggression in a dosedependent fashion without disrupting general movement. Infusing SCH‐23390 or sulpiride did not disrupt movement in the open‐field tests at dosages reducing operant responding in the previous aggression tests.

Conclusions: Our results suggest that both D1‐like and D2‐like DA receptors in the ventral striatum may mediate the rewarding properties of aggression as a positively reinforcing stimulus.
Anxiolytic‐like effect of the PDE4 inhibitor rolipram and its relationship to cAMP signaling and hippocampal neurogenesis
Authors: Li Yun‐Feng , Huang Ying , Stolinski Lisa R , O'Donnell James M , Zhang Han‐Ting
Published: June 2021

Phosphodiesterase‐4 (PDE4), an enzyme that hydrolyzes cyclic AMP (cAMP), plays a critical role in controlling intracellular cAMP concentrations. Since cAMP signaling regulates anxiety, it was of interest to know if PDE4 was involved in anxiety‐like behavior. In behavioral tests sensitive to anxiolytic drugs, including elevated‐plus maze, light‐dark transition, and holeboard tests, chronic treatment with the PDE4 inhibitor rolipram (1.25 mg/kg, 17–21 d) produced anxiolytic‐like effects in mice and increases in cAMP and phospho‐CREB (pCREB) in both the hippocampus and prefrontal cortex and hippocampal neurogenesis (i.e., increased BrdU‐positive cells in the dentate gyrus). In addition, the effects of rolipram on hippocampal pCREB and neurogenesis were concurrently blocked by methylazoxymethanol (MAM, 5 mg/kg, 14 d), a toxin to proliferation cells; its anxiolytic‐like effect also was attenuated by MAM in a parallel pattern. Further, the inhibitory effects of MAM disappeared 19 d after MAM withdrawal. Finally, the effects of rolipram and/or MAM on BrdU‐labeled cells, pCREB, and behavior were highly correlated with each other. These results suggest that 1) PDE4 is involved in the regulation of anxiety‐like behavior; 2) cAMP/CREB signaling plays an important role in anxiolytic‐like and neurogenic effects of rolipram; 3) rolipram‐induced neurogenesis contributes to its anxiolytic activity.
Differential role of CREB in novelty‐seeking behavior by adolescent and adult rats
Authors: Philpot Rex M. , Wecker Lynn
Published: June 2021

Novelty‐seeking behavior by both adolescents and adults may be classified as high (HR) or low (LR) responding, with adolescents exhibiting higher activity than adults. In adults, cAMP‐response element binding protein (CREB) is associated with behavioral reactivity to novel stimuli, but its role in adolescence, when cAMP signaling is tempered, is unknown. To determine whether differences exist between adults and adolescents, CREB and pCREB from prefrontal cortex and ventral striatum from adolescent and adult rats were measured one week following exposure to a novel environment. Adolescents moved significantly faster and farther than adults in the novel environment with 16–18% exhibiting activity above the highest activity manifest by adults, and there was a greater percentage of the HR phenotype in adolescents relative to adults. Cellular analyses indicated that LR adults had 2‐fold greater CREB and pCREB than LR adolescents, whereas HR adults had 50% the CREB and pCREB of HR adolescents. In addition, CREB and pCREB were 1.5 times greater in HR than LR adolescents, whereas CREB and pCREB were 50% lower in HR than LR adults. Results suggest that CREB expression and activity exhibit distinct alterations associated with novelty‐seeking in adolescents relative to adults and may mediate age‐dependent differences in the phenotypic expression of novelty–seeking behavior. (Supported by NIAAA grant AA016449.)
Smooth muscle specific knockdown of L‐type calcium channel by exogenous microRNA (miRNA)
Authors: Pang Li , Stimers Joseph , Rhee Sung , Palade Philip
Published: June 2021

In spontaneously hypertensive rats, vascular L‐type calcium channel pore forming α1 subunit (Cav1.2) protein expression, L‐type calcium current (CaL) and vascular tone are all increased, due to increased expression of the ubiquitously expressed isoform containing an exon 1b encoded N‐terminus. The objective of this study is to develop shRNA or miRNA constructs against the non‐cardiac form of Cav1.2 (exon1b isoform) to reverse the upregulation in a tissue‐specific manner. Cav1.2 exon 1b‐specific shRNA, miRNA, and appropriate controls were constructed and tested for silencing effects. In HEK cells, rat Cav1.2 expression vector (rCav1.2) was cotransfected with different constructs, CMV promoter‐driven miRNA (Cav1.2) had the strongest effect and decreased rCav1.2 protein ∼72%. More interestingly, miRNA(Cav1.2) driven by the vascular smooth muscle‐specific promoter SM22α reduced endogenous Cav1.2 expression (western blot) ∼51% and decreased CaL (barium) current (patch clamp) ∼46% in transfected A7r5 cells. Moreover, the SM22‐GFP‐miRNA(Cav1.2) had no effect on cotransfected rCav1.2 expression in HEK cells and showed little or no GFP expression in PC12 or HL1 cells.

Conclusion:

SM22–GFP‐miRNA(Cav1.2) can effectively and smooth muscle‐specifically reduce Cav1.2 expression. Coupled to adeno‐associated virus, this may provide a novel long‐term therapy for hypertension. Supported by NIH HL63903.
Endothelial dysfunction in pulmonary arteries from diabetic rats
Authors: Lopez‐Lopez JG , Moral J , Frazziano G , Gómez‐Villalobos MJ , Flores‐Hernández JV , Flores G , Gutiérrez‐Godinez J , Perez‐Vizcaino F
Published: June 2021

Diabetes mellitus has been recently associated to a higher prevalence of pulmonary hypertension. Diabetes leads to multiple cardiovascular alterations, including endothelial dysfunction in multiple vascular beds. However, the effects of diabetes on the pulmonary circulation are unknown. We analyzed the endothelial function in isolated pulmonary arteries from rats with streptozocin‐induced diabetes. Diabetes led to endothelial dysfunction as denoted by a marked decrease in the endothelium‐dependent vasodilatation induced by acetylcholine in pulmonary arteries from streptozocin‐treated compared to control rats. Acetylcholine‐induced relaxations were abolished by the NO synthase inhibitor L‐NAME. The contractile responses to L‐NAME were also reduced in arteries from diabetic rats. However, the endothelium‐independent vasodilatation to sodium nitroprusside was similar in both groups. The endothelial dysfunction was reverted by incubating the arteries with superoxide dismutase or the membrane NADPH oxidase inhibitor apocynin. Endothelial dysfunction was associated with an increase the production of superoxide as measured by dihydrotheidium and an increased expression of the NADPH oxidase subunit p47phox. In conclusion, we show for the first time that diabetes induced endothelial dysfunction in pulmonary arteries due to enhanced NADPH oxidase‐derived superoxide production
An aqueous extract of Coptis chinensis (ACC) augments rat penile tissue erection
Authors: Lee Jun Kyoung , Yang Sun Nye , Kang Dae Gill , Li Xiang , Lee Ho Sub
Published: June 2021

The aim of the present study was to investigate whether an aqueous extract of Coptis chinensis (ACC) augments penile erection in vivo and in ex vivo. In isolated endothelium‐intact rabbit corpus cavernosum preconstricted with phenylephrine, ACC (10−7−10−4 g/ml) relaxed penile smooth muscle in a dose‐dependent manner, which was inhibited by pretreatment with a nitric oxide synthase inhibitor, and a soluble guanylyl cyclase (sGC) inhibitor, respectively. In addition, incubation of endothelium intact rabbit corpus cavernosum tissues with ACC increased cGMP production in a dose‐dependent manner. ACC‐induced cGMP production was not blocked by pretreatment with L‐NAME or ODQ. Moreover, IBMX, a non‐selective phosphodiesterase (PDE) inhibitor, or zaprinast, a type 4, 5‐selective PDE inhibitor caused more increase in cGMP production than ACC alone. On the other hand, there are no significant difference between increases in penile tissue cGMP production induced ACC and co‐treated with ACC and IBMX or zaprinast, respectively. ACC (100, 500 mg/kg) also caused increase in intracavernous pressure and area under the curve in rats in a dose dependent manner. These results suggest that ACC maybe induce penile erection through the activation of penile tissue NO/cGMP system and/or inhibition of PDE in rat corpus cavernosum.
Development of tools to assess visceral adipose tissue (VAT) accumulation during the development of erectile dysfunction (ED) and during pharmacotherapy
Authors: Komolova Marina , Hannan Johanna L , Maio Tina , Blazer Mark C , Shamloul Rany M , Adam Michael A
Published: June 2021

ED is a multi‐factorial process involving cardiovascular, metabolic and hormonal factors. In particular, obesity is in epidemic proportions, with the incidence of ED in obese men reaching ~80%. Despite this, few animal models have mechanistically linked ED and obesity, and none have yet provided a testing ground for new pharmacotherapeutic strategies in obesity‐related ED. The present study was performed to begin to address this gap. Erectile responses were induced using the dopaminergic agonist, apomorphine (80 ug/kg, s.c.), in aging (15–75 wks) Sprague‐Dawley and Wistar rats. Assessment of physical properties included: body weight (BW), body length (BL), waist circumference (WC), and post‐mortem VAT mass. MR imaging was also used to assess adiposity vs. post‐mortem data. Erections declined in an age‐related (20–75 wks) manner, paralleling increases in BW and 7‐fold increases in VAT. Regression analysis revealed the best non‐invasive indicators of VAT by rank order of R2 were: BW:BL ratio > WC >> BW. These studies demonstrate the importance of assessing age‐related changes in physical features and adiposity (particularly VAT), with respect to changes in penile vascular responses. Previous studies have linked recovery of erectile responses with weight loss; whether this improvement in function is specifically linked to reductions in VAT is currently being studied. (Funds: Heart and Stroke Foundation)
Homotropic and heterotropic cooperativity of CYP3A4 and drug‐drug interactions
Authors: Denisov Ilia G , Frank Daniel , Grinkova Yelena V , Sligar Stephen G
Published: June 2021

The allosteric behaviour of monomeric human cytochrome P450 CYP3A4 solubilized in nanoscale lipid bilayers was systematically studied in order to understand the mechanisms of homotropic and heterotropic cooperativity. The ability of CYP3A4 to simultaneously bind multiple drug molecules alters their metabolism and gives rise to drug‐drug interactions. Using global analysis of several data sets obtained in different experiments under identical conditions, we are able to represent the overall cooperative properties of CYP3A4 and the functional contributions of each binding intermediate. We show that the homotropic cooperativity observed in testosterone (TS) hydroxylation is due to negligible product formation when only one substrate molecule is bound, and not to any significant conditional interaction free energies of substrate binding. A two‐dimensional binding isotherm is obtained for the heterotropic interactions between TS and alpha‐naphthoflavone, allowing complete deconvolution of homotropic and heterotropic cooperativity in this important system. . Supported by NIH grants GM 33775 and GM 31756 to S.G.Sligar.
Dual targeted molecular imaging for atherosclerotic plaque detection
Authors: Ferrante Elisa A , Rychak Joshua J. , Klibanov Alexander , Ley Klaus
Published: June 2021

Selectins and VCAM‐1 are responsible for leukocyte adhesion in atherosclerosis and have been proposed as targets for molecular imaging of the disease. Currently, no method exists to reliably detect inflamed atherosclerotic plaque. In this study, perfluorocarbon‐filled microbubble contrast agents are coupled by a biotin‐streptavidin bridge with mAb MVCAM.A(429), a sialyl Lewisx polymer (sLex) or both (dual). Adhesion of these agents respectively to recombinant mouse VCAM‐1 and P‐selectin substrates was investigated in a parallel plate flow chamber at wall shear stresses between 0.5 and 4.5 dyne/cm2. The amount of antibody on the bubbles was determined by flow cytometry. The amount of P‐selectin and VCAM‐1 on substrates was determined with 125I radiolabeled antibodies. Accumulation of both MVCAM.A(429) or sLex microbubbles was higher on P‐selectin or VCAM‐1 coated substrates rather than casein blocked controls. Both dual targeted and MVCAM.A(429) microbubbles showed increased adhesion over controls. Interactions between SLex bubbles and the substrate predominantly consisted of rolling rather than adhesion. Dual targeting can increase the binding efficiency of contrast agents to substrates that mimic inflamed endothelium, suggesting they may be useful for atherosclerotic plaque detection. Supported in part by NIH 2 R01 EB002185.
Modification of a calcein‐based assay for monitoring proteolytic activity in tissue suffusate
Authors: Mikel Kyle , Bingaman Susan , Huxley Virginia H.
Published: June 2021

This investigation was aimed at developing an assay to characterize protein degradation in effluent collected from tissues on which microvascular permeability studies were being conducted (Bingaman S., FASEBJ: 20, 2006). In particular, we were measuring protein clearance from venules and assaying total protein in the effluent. The assumption we wanted to test was whether the protein levels reflected intact material, whether proteolysis was occurring, and whether the treatments which altered protein clearance reflected changes in proeolytic activity. The approach we used was based on the quenching of calcein (Ex 450 nm, Em 530 nm) by Cu(II) and the ability of proteolytic products (amino acids and peptides) to coordinate with the Cu(II), removing it from the calcein, allowing it to fluoresce (Dean, K., Biorganic & Medicinal Chem Letters, 2003). After optimization of assay conditions, samples collected from representative leak index experiments (Bingaman S., FASEBJ: 20, 2006) were tested. The results showed a clear difference between samples with respect to the distribution of proteins and small peptides under both control and test conditions. Therefore, it can be concluded that this assay can be used to screen for proteolytic activity in the microvasculature.

Supported by NIH HLO78816, NASA NN04GA50G, AHA 0615515Z (RS), NIHCO6RRO17353 and Missouri Academy
Proteomic analysis of connective tissue growth factor activation in normal and scleroderma lung fibroblasts
Authors: Bogatkevich Galina S. , Ludwicka‐Bradley Anna , Singleton C. Beth , Silver Richard M.
Published: June 2021

Connective tissue growth factor (CTGF, CCN2) is over expressed in lung fibroblasts isolated from patients with interstitial lung disease (ILD) and is considered to be a molecular marker of fibrosis. To understand the significance of elevated CTGF we investigated the changes in lung fibroblast proteome in response to CTGF overexpression. Using two‐dimensional gel electrophoresis (2‐DE) followed by in‐gel proteolytic digestion and mass spectrometric analysis, we identified 13 proteins affected by CTGF. Several of the CTGF‐induced proteins, such as pro‐α (I) collagen and cytoskeletal proteins vinculin, moesin, and ezrin, are known to be elevated in pulmonary fibrosis, whereas nine of 13 proteins have not been studied in pulmonary fibrosis and are, therefore, new ILD targets for CTGF. Our study demonstrates that one of the novel CTGF‐induced proteins, IQ motif containing GTPase activating protein (IQGAP)‐1, is elevated in lung fibroblasts isolated from scleroderma patients with ILD. Scleroderma lung fibroblasts and CTGF‐treated normal lung fibroblasts are characterized by increased collagen synthesis, over‐expression of alpha‐smooth muscle actin, increased collagen gel contraction, and increased rate of migration in a wound healing assay. Depletion of IQGAP‐1 expression by small interfering (si)RNA inhibited CTGF‐induced migration of normal lung fibroblasts and significantly decreased the rate of migration of scleroderma lung fibroblasts. These findings further implicate the importance of CTGF in lung tissue repair and fibrosis and suggest that IQGAP‐1 mediates the migration of lung fibroblasts to the damaged tissue.
Estrogen reduces inflammation of asthmatic airways by inhibiting pathways leading to oxidant stress.
Authors: Dimitropoulou Christiana , Snead Connie , Hobbs Janet , Pollock Jennifer S , Catravas John D
Published: June 2021

We have previously reported that estrogen replacement therapy reduces inflammation in a murine model of allergic asthma. We now studied the mechanisms responsible for this protective effect. Female C57/Bl6 mice were ovariectomized, implanted with subcutaneous osmotic minipumps releasing either vehicle or estrogen and subjected to a 25‐day regimen of ovalbumin sensitization. On day 26, animals were sacrificed and lung tissue was analyzed as described below. Asthmatic, vehicle‐treated mice exhibited strong oxidant stress, as revealed by immunohistological staining of lung tissue for 8‐hydroxy‐2‐deoxyguanosine (8‐OHdG), an indicator of oxidative DNA damage; 8‐OHdG staining was significantly reduced in asthmatic, estrogen‐treated mice and resembled that in non‐asthmatic controls. Furthermore, asthmatic, vehicle treated animals demonstrated increased Akt phosphorylation, and Rac‐1 activation, suggesting that NADPH oxidase may be an important contributor to the observed oxidant stress in asthmatic animals. Akt phosphorylation and Rac‐1 activation were profoundly inhibited in estrogen‐treated asthmatic mice, to levels resembling those on non‐asthmatic controls. These data suggest that the protective effects of estrogen replacement therapy may include inhibition of pathways leading to increased oxidant stress.
The Hydrophobic Surfactant Proteins Destabilize Pulmonary Surfactant Monolayers
Authors: Hall Stephen B. , Lhert Florence , Yan Wenfei , Biswas Samares C.
Published: June 2021

To determine if the hydrophobic surfactant proteins affect the stability of pulmonary surfactant monolayers at an air/water interface, we compared the kinetics of collapse for the complete set of lipids in calf surfactant with and without the proteins. Monomolecular films spread at the surface of captive bubbles were compressed at 37°C to surface pressures (π) above 46 mN/m, at which collapsed first occurred. The rate of area‐compression required to maintain constant π determined the rate of collapse. For films with and without the proteins, higher π initially produced faster collapse, but the rates then reached a maximum and decreased to minimal values. The maximum rate for the lipids with the proteins was almost twice the value for the lipids alone. Because small changes in π produced large shifts in the rate close to the fastest collapse, compressions at constant rates also established the threshold speed required to achieve high π as an indirect measure of the fastest collapse. Both samples produced a sharply defined threshold that occurred at slightly faster compression with the proteins present, supporting the direct measurements that the proteins produce a faster maximum rate of collapse. At 47–53 mN/m, the hydrophobic surfactant proteins destabilize the compressed monolayers, and tend to limit access to the higher π at which the lipid films become metastable.
Dilution potential (DP) across rat alveolar epithelial cell monolayers
Authors: Zhang Yifan , Zhang Lequan , Borok Zea , Crandall Edward D. , Kim Kwang‐Jin
Published: June 2021

In this study, we estimated permeability ratio (PNa/PCl) between Na and Cl for paracellular pathways of primary rat alveolar epithelial cell monolayers (RAECM). PNa/PCl was estimated using Goldman‐Hodgkin‐Katz equation and DP measured across RAECM at 5–7 days. To inhibit Na pump activities, RAECM were bathed on both sides with K‐free Ringer's solution (KFRS, in which 5 mM KCl is replaced by equimolar NaCl). DP was measured after lowering apical [NaCl] tenfold by replacing apical fluid with a 9:1 mixture of isotonic K‐free mannitol solution and KFRS. Osmolarity of all solutions was adjusted to 300 mOsm with mannitol. DP was further corrected for liquid junction potential of voltage‐sensing electrodes across blank filters. Results show that equivalent short‐circuit current across RAECM bathed on both sides with KFRS is not significantly different from zero with electrical resistance >1,000 Ωcm2. When apical [NaCl] was lowered tenfold, corrected DP ∼ −13 mV (apical as reference) was observed, yielding PNa/PCl ∼1.8. For comparison, the free solution diffusivity ratio of Na to Cl is ∼0.7, while paracellular PNa/PCl of various epithelial barriers ranges from ∼0.2 and ∼0.5 for frog skin and bullfrog lung to ∼1.3 and ∼10 for rabbit proximal tubule and rat jejunum, respectively. These data indicate that passive ion conductance through tight junctions of RAECM is greater for Na than Cl. (Support: NIH and Hastings Foundation.)
Transforming Growth Factor‐β1 Inhibits CFTR‐dependent cAMP‐stimulated Alveolar Epithelial Fluid Transport: Critical Role of Interleukin‐8
Authors: Roux Jeremie , Ganter Michael T , Goolaerts Arnaud , Finkbeiner Walter , Matthay Michael A , Pittet Jean‐Francois
Published: June 2021

β‐adrenergic agonist‐dependent stimulation of the lung fluid clearance is an important mechanism that protects the lung from alveolar flooding. In this study we hypothesized that critical mediators of acute lung injury (ALI), such as transforming growth factor‐β1 (TGF‐β1) and interleukin‐8 (IL‐8), could directly antagonize the epithelial response to β‐adrenergic agonists. Short circuit current experiments revealed that TGF‐β1 inhibits CFTR‐specific β‐agonist‐stimulated vectorial Cl‐transport across the apical membrane of primary rat and human alveolar epithelial type II (ATII). TGF‐β1 also significantly decreased β‐agonist‐stimulated cAMP production resulting in the inhibition of the CFTR promoter activity and gene expression in ATII cells. We found that this TGF‐β1‐dependent inhibition required IL‐8 and was mediated by desensitization the β‐adrenergic receptor through both TGF‐β1 and IL‐8 signaling pathways. Consistent with the in vitro results, we showed that TGF‐β1 requires IL‐8 to inhibit the β‐agonist‐stimulated fluid transport across the distal airspace epithelium in vivo in rats. In summary, the results show for the first time that TGF‐β1 and IL‐8 inhibit the stimulated vectorial chloride transport across the distal lung epithelium, thus impairing the β‐adrenergic‐dependent protective mechanism against alveolar flooding.

NIH P50HL074005 (JFP), ALA Award and T32 GM008440 (JR)
Lipopolysaccharide (LPS)‐induced effects on Na+ transport and inflammatory response‐gene transcription in the guinea‐pig airway epithelium
Authors: Dodrill Michael , Rao K. Murali Krishna , Meighan Terence , Fedan Jeffrey S.
Published: June 2021

Earlier, we reported that systemic administration of LPS hyperpolarizes transepithelial potential difference (Vt). The increase in Vt was abolished by amiloride and inhibited by indomethacin. These findings suggested an increase in inflammatory‐mediator regulated Na+ transport. We investigated the effects of LPS on the mRNA levels of the epithelial sodium channel α subunit (αENaC), cyclooxygenase (COX) 2, endothelial nitric oxide synthase (eNOS), interleukin (IL)‐1β, inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)α, which regulate ENaC activity and turnover, using real‐time RT‐PCR. Guinea pigs received 4 mg/kg LPS (i.p.); epithelial cells (EC) and alveolar macrophages (AM) were examined after 3 or 18 h. EC COX2 increased by 36±26‐fold at 3 h and was elevated by 15±12‐fold at 18 h. In EC, TNFα, IL‐1β and iNOS were increased at 3 h (32±17‐, 25±12‐ and 7±5‐fold, respectively) and decreased to baseline after 18 h. AM COX2 was elevated at 3 (163±112‐fold) and 18 h (8±7‐fold). AM IL‐1β and AM TNFα were elevated at 3 h (127±60‐ and 20±7‐fold, respectively), and returned to baseline after 18 h. In contrast, AM eNOS increased by 4.7±5.4‐fold only at 18 h. The other transcripts were not increased more than 4‐fold in either tissue at 3 or 18 h. Thus, αENaC mRNA was not affected by LPS. An increase in ENaC transcription cannot explain the LPS‐induced increase in Vt.

Funded by NIOSH.
Adaptive Changes of Sodium and Potassium Transporters in CRF Rat Kidneys
Authors: Kim Sejoong , Heo Nam Ju , Jeong Ji Yong , Oh Yun Kyu , Na Ki Young , Joo Kwon Wook , Han Jin Suk
Published: June 2021

In chronic renal failure (CRF), residual nephrons can magnify their excretion of sodium and potassium. However, long‐term adaptive mechanism of renal tubular transporters in CRF has not been fully evaluated. We performed immunoblotting and immunocytochemistry, thus investigating the sequential changes of expression of major renal sodium and potassium transporters in rats from 4 weeks to 12 weeks after 5/6 nephrectomy (Nx). At 4 weeks, the density of NKCC2, NCC, ENaC‐α, and ENaC‐γ in CRF rats significantly increased (477%, 222%, 451%, and 435% of control, respectively) although the expression of NHE3 and SGLT1 did not altered. In contrast, expression of NKCC2 and NCC markedly decreased at 12 weeks (55.4% and 30.8%, respectively), and NHE3 and SGLT1 also decreased at 12 weeks (48.4% and 24.6%, respectively). The protein abundance of ENaC‐α significantly increased during the whole period. Urinary potassium excretion rate was well‐correlated with urinary urea or sodium excretion rate, although immunoblotting of ROMK and Na‐K‐ATPase showed a gradual decrease in the late stages of CRF. We suggest that this sequential decrease of major renal sodium transporters may be a main mechanism for increased urinary sodium excretion, and that increased urea excretion rates or distal sodium delivery may be a major explanation for increased urinary potassium excretion in CRF rats.
The effect of PPARγ Activation on the Expression of Renal Proximal Tubule Sodium Transporters NHE3 and Na,K ATPase in vivo and in vitro
Authors: Dobrian Anca Dana , Ambrozewicz Marta A , Sekulovich Miroslav , DeBose Sophia Chung
Published: June 2021

A rat model of obesity and hypertension was used for in vivo studies and renal proximal tubule epithelial cells (RPTEC) for the in vitro studies. mRNA expression was determined by quantitative real time PCR and protein expression by semi‐quantitative Western blotting in membrane fractions from cells or renal cortex. In obese rats, the mRNA and protein expression of NHE3 are both increased compared to lean rats, while the mRNA and protein expression of Na,K ATPase are not different between the two groups. Pioglitazone treatment for 2 weeks decreased both mRNA and protein expression of NHE3 in obese and lean rats. The treatment also decreased mRNA and protein expression of Na,K ATPase in obese but not the lean rat group. To determine if pioglitazone exerts its effect via the PPARγ receptor, RPTEC were transiently transfected to overexpress PPARγ or treated with PPARγ siRNA. In cells overexpressing PPARγ the effect was identical with control cells. However, in cells treated to PPARγ siRNA, the effect of pioglitazone on both transporters expression was blunted. In conclusion: pioglitazone acts to reduce both transcription and protein synthesis of NHE3 regardless of the metabolic context, but exerted it's effect on Na,K ATPase only in obese hypertensive animals. The in vitro studies indicate that the effect of the drug is mediated via a PPARγ dependent mechanism.
PKC activation downregulates the human Na+/L‐ascorbic acid transporter SVCT1 via its derecruitment from the plasma membrane
Authors: Shawki Ali , Illing Anthony C , Worrell Roger T , Mackenzie Bryan
Published: June 2021

Intestinal absorption and renal reabsorption of vitamin C (L‐ascorbic acid, L‐Asc) is mediated by the epithelial Na+/L‐Asc cotransporter SVCT1 (SLC23A1). L‐Asc transport is downregulated in Caco‐2 cells after pretreatment with ascorbic acid (MacDonald et al [2002] Br J Nutr 87, 97) suggesting that high‐dose supplements are not an efficient means of increasing the body pool of vitamin C, and highlighting the need to understand the regulation of SVCT1. Since pro‐oxidant environments potently inactivate protein kinase C (PKC), we examined a possible role for PKC in regulating SVCT1, using radiotracer and voltage‐clamp techniques in Xenopus oocytes expressing SVCT1. Uptake of 100 μM L‐[14C]Asc (100 mM Na+, pH 7.5) was inhibited 91% ± 11% by the PKC activator phorbol 12‐myristate 13‐acetate (PMA, 100 nM) (P < 0.001) but not by its inactive analogue 4αPMA (P = 0.27). The PMA effect was blocked by the PKC inhibitors Gö6850 and Gö6976, implicating a conventional‐type PKC isozyme. PMA treatment resulted in internalization of SVCT1 protein since a 94% ± 7% reduction in L‐Asc‐evoked currents was associated with (1) a decrease in presteady‐state charge (∝ the number of functional units in the plasma membrane), (2) a decrease in membrane capacitance (∝ surface area), and (3) an increase in cell resistance. PMA reduced the Vmax without effect on the K0.5 for L‐Asc. Our data support a role for a conventional PKC isozyme in downregulating SVCT1 via derecruitment of SVCT1 from the plasma membrane.
Cloning of Xenopus laevis and Strongylocentrotus purpuratus Na‐K‐2Cl cotransporters
Authors: Gagnon Kenneth B. , England Roger , Delpire Eric
Published: June 2021

The Na‐K‐2Cl cotransporter (NKCC1) mediates the movement of ions and water across epithelia, and is important in the maintenance of cell volume. Our laboratory has used the amphibian (Xenopus laevis ‐ African clawed frog) oocyte as a heterologous expression system for the study of mammalian NKCC1 function. The release of the echinoderm (Strongylocentrotus purpuratus ‐ purple sea urchin) genome, which was lacking all NKCC1 regulatory elements (two SPAK binding domains, the PP1 binding domain, and the six conserved threonine residues) in its amino terminus, provided a unique opportunity for us to compare the molecular differences between echinoderm, amphibian, and mammalian cotransporters. In this study, we cloned NKCC1 from RNA extracted from the frog oocyte and sea urchin gonadal tissue. We found that the amino terminal domain of frog NKCC1, although smaller than mouse by 48 amino acid residues, has two SPAK binding domains and all six threonine residues previously identified as important to NKCC1 function. The sea urchin NKCC1 amino terminal domain, misidentified by the genome project, has only 149 residues compared to the 278 residues of mouse NKCC1, one SPAK binding domain and two of the six conserved threonine residues (sea urchin T87/T92 homologous to mouse T206/T211). The cloning of NKCC1 from these two new species allow us to further address the role of the N‐terminus in the regulation of the cotransporter.
Interaction between P2Y receptors and TRPV1 on kidney specific sensory neurons
Authors: Wang Hui , Wang Donna H. , Galligan James J.
Published: June 2021

The transient receptor potential vanilloid 1 (TRPV1) receptor is a ligand‐gated cation channel expressed primarilyin sensory nerves and functions as a molecular integrator of sensory input. TRPV1 expressed by sensory neurons also detects salt‐induced changes in tissues and regulates salt sensitivity in hypertension. Sympathetic nerves are closely aligned with sensory nerves in the renal vasculature and sympathetic nerves release ATP that can act on P2Y receptors expressed by sensory nerve fibers. We studied the interaction between P2Y receptors and TRPV1 in kidney specific sensory neurons which are critical for salt balance. Application of Fast Blue (FB) to the nerves surrounding renal artery retrogradely labeled neurons in dorsal root ganglia (DRG, T12‐L4) of rats. Ganglia were harvested and placed in tissue culture for whole cell patch clamp recording from FB‐labeled, kidney‐specific sensory neurons. ATP pretreatment reversed capsaicin (CAP)‐induced TRVP1 desensitization. UTP, a P2Y2 and P2Y4 receptor agonist, also reversed CAP‐induced TRPV1 desensitization. Immunocytochemical studies showed that P2Y1 and P2Y2 receptors were co‐expressed with TRPV1 on FB‐labeled neurons. These data indicate that P2Y2/P2Y4 receptors may function to modulate TRPV1 activity. This may be a mechanism of interaction between sympathetic and sensory nerves supplying the renal vasculature.
Molecular Modeling of Acid Sensing Ion Channels
Authors: Qadri Yawar J , Fuller Catherine M , Benos Dale J
Published: June 2021

Acid sensing ion channels (ASIC) proteins are a subfamily of the Degenerin/Epithelial Sodium Channel (ENaC) superfamily of proteins. These proteins interact with themselves and other family members to form diverse ion channels implicated in numerous physiological and pathophysiological processes such as hypertension and nociception and are all inhibited by amiloride. Recently, the first crystal structure of a galline member of this family was obtained, allowing a look at the exact structure and arrangement of the channel complex. However, functional and pharmacological data of chicken ion channels are limited. Using MODELLER 9v2, homology models of the better characterized huASIC‐1b, huASIC‐2b, and αβγ‐huENaC channels were derived. While both ASIC‐1 and ASIC‐2 models appear similar to the template structure, models of ENaC channels diverge with several undefined domains, suggesting that ENaCs may be structurally different from ASICs. Regardless, with models in hand, it is now possible to perform in silico screening to find novel small molecule inhibitors. Preliminary inhibitor docking studies for amiloride are consistent with sites described in the literature as critical for amiloride inhibition. Further work will use molecular dynamics to better optimize the models and to further examine inhibitor interactions with ASIC/ENaC channels. This study was supported by NIH Grant CA101952 and DK37206.
Histamine induces sonic hedgehog secretion across the apical membrane of polarized parietal cells
Authors: Zavros Yana , Orr Melissa A. , Malinowska Danuta H.
Published: June 2021

Objective:

Sonic Hedgehog (Shh) is processed and secreted from parietal cells by a unique acid‐ and protease‐dependent mechanism (Zavros et al., 2007, JBC). The current study seeks to determine the cellular location of Shh within the gastric parietal cell.

Methods:

Shh expression was studied using cimetidine (resting) or histamine (stimulated)‐treated rabbit gastric glands and immunoisolated tubulovesicles. Apical (AP) and basolateral (BL) Shh secretion was studied using polarized human gastric cancer cells (23132/87).

Results:

Confocal microscopy of gastric glands triple stained for Shh, H+,K+‐ATPase and zymogen or neck cell markers showed expression of Shh within the canaliculus of stimulated parietal cells. Western blots showed Shh and H+,K+‐ATPase expression in resting immunoisolated tubulovesicles. While the Shh precursor was detected in resting membranes, increased expression of processed Shh was observed in stimulated tubulovesicles. Surface biotinylation of polarized 23132/87 cells revealed expression of the Shh precursor at the AP membrane and processed Shh secreted in the AP media. Shh was not detected in either the BL biotinylated membrane lysates or media.

Conclusion:

Shh is expressed in H+,K+‐ATPase‐containing membranes suggesting that processing and secretion occurs across the apical membrane of parietal cells.
Protein expression and distribution of murine calcium‐activated chloride channels 1 and 2 (mCLCA1/2) in epithelia of the gastrointestinal (GI) tract and kidney (Ki)
Authors: Thevenod Frank , Wittschen Petra , Torchalski Blazej , Gruber Achim D.
Published: June 2021

Calcium‐activated chloride channels (CLCA) have been implicated as anion channels, channel regulators and cystic fibrosis phenotype modulators, but their function remains unclear. We generated a polyclonal antibody (pAb 849) against a peptide that is common to mCLCA1/2, has ~70% aa identity with mCLCA3/4, but none with mCLCA5/6. pAb 849 detected mCLCA1 expressed in HEK293 by immunoblotting (IB) and immunoprecipitation, where 125 and 90 kDa bands suggest posttranslational modification. Flag‐tagging of mCLCA1 yielded only a 130 kDa band. pAb 849 cross‐reacted with mCLCA3 in IB with bands at 120, 100 and 75 kDa. Using immunohistochemistry and comparison with mCLCA3 (pAb p3a), mCLCA1/2 specific labelling was found in pancreas acini (apical (AP), zymogen granules (ZG)), but not in ducts. pAb 849 IB of ZG membranes yielded a 130 kDa band. Specific signals were also seen in small ducts of seromucous salivary glands (AP, intracellular (IC)) but not in excretory ducts or acini. Stomach parietal (AP, IC) and jejunal crypt cells (AP) were specifically labelled. Signals in stomach mucous and jejunal goblet cells may be due to mCLCA3 cross‐reactivity. Only AP areas of Ki distal tubules were stained. mCLCA1/2 positive tissues do not express mCLCA4, therefore the data indicate mCLCA1/2 expression in AP membranes of GI and Ki and ZG membranes, but a function as secretory proteins cannot be excluded.

Funding: German CFF F04/04
Selective Estrogen Receptor Agonists Enhance Blood Pressure, Thromboxane and Prostacyclin in Aortic Coarctation‐Induced Hypertensive Female Rats
Authors: Sellers Minga , Xu Feng , Stallone John
Published: June 2021

Previously, we reported that estrogen receptors (ERα, ERβ) exert differing effects on mesenteric arterial reactivity to vasopressin (VP) and that estrogen enhances constrictor prostanoids by upregulating thromboxane synthase and cyclooxygenase‐2 message in rat aorta. We measured mean arterial blood pressure (BP), thromboxane (TXA2) and prostacyclin (PGI2) release by mesenteric arterioles (200 μm) in normo‐ and aortic coarctation‐induced hypertensive (NT and HT) Sprague‐Dawley female rats that were intact (INT), ovariectomized (OvX), or OvX + treated with ER‐selective agonists DPN (ERβ) or PPT (ERα), 100μg/rat/day (13–15 days). BP in PPT HT (180±4 mmHg) is similar to INT HT (183±4) and DPN HT (182±4). BP is lowest in OVX HT (153±3). BP in NT did not differ (mean = 127±1). Basal TXA2 was lowest in OVX and DPN NT (51 ± 20; 40 ± 7 pg/mg tissue/45 min), while INT or PPT NT are higher (93 ± 1; 93 ± 10). HT increases basal TXA2 threefold. VP (10−8) increases TXA2 least in DPN NT (79 ± 11) vs INT NT (108 ± 1), OVX NT (137 ± 33) or PPT NT (134 ± 31). HT increases VP stimulated TXA2. PPT HT increased the most (643 ± 137) vs INT HT (415 ± 46), OVX HT (277 ± 56) or DPN HT (334 ± 33). PGI2 shows similar trends in all groups; PPT enhanced basal and VP stimulated PGI2 the most. These data suggest that ERα enhances TXA2 and PGI2 to a greater extent than ERβ. The increased PGI2 does not afford protection to BP in PPT HT. (Supported by NIH: HL‐080402)
Opposing effects of gonadal hormones and the sex chromosome complement on renal 11β hydroxysteroid dehydrogenase (11β HSD‐2) protein abundance and angiotensin‐induced hypertension
Authors: Ecelbarger Carolyn M. , Tiwari Swasti , Shi Min , Packer Randall K. , Arnold Arthur , Sandberg Kathryn , Roesch Darren
Published: June 2021

The effects of gonadal hormones (GH) on angiotensin II (Ang II)‐induced hypertension (HT) have been widely studied, but little is known about the independent effects of the sex chromosome complement (SCC). HT is attenuated in young female (F) mice as compared to males (M). Reduced activity of the renal enzyme, 11β HSD‐2, is associated with altered corticosteroid activity and increased HT. We hypothesized GH and SCC may regulate renal 11β HSD‐2 activity and contribute to sex differences in Ang II‐induced HT. M and F C57BL mice were infused with vehicle (C) or Ang II (A, 800 ng/kg·bw/min) for 10 days (n = 6/group). Western blotting revealed that Ang II infusion and M sex significantly reduced 11β HSD‐2 protein (2‐way ANOVA for treatment, p = 0.034 and sex, p = 0.003). Band densities were (% MC): 100 ± 10 (MC); 140 ± 12 (FC); 84 ± 7 (MA); 109 ± 7 (FA). To determine the role of the SCC (XX or XY) independent of GH, we also conducted experiments in Sry transgenic mice whose phenotypic sex is separated from SCC by translocation of the testes‐determining gene, Sry onto an autosome. In these ovariectomized (OVX) and castrated (CAS) mice, the XX SCC was associated with significantly greater HT (153 vs. 144 mm Hg with Ang II infusion) and significantly reduced renal 11β HSD‐2 abundance. Band densities were (% Ovx XX): 100 ± 8 (Ovx XX); 122 ± 7 (Ovx XY); 96 ± 9 (Cas XX); 121 ± 11 (Cas XY). Thus, young, intact F mice have increased 11β HSD‐2 levels due to actions of GH that oppose the SCC. The HT‐promoting effects of the XX SCC may become more apparent with age as GH levels decrease. NIH‐support
Effect of estrogen on sympathetic nervous system activation in high salt hypertension
Authors: Hinojosa‐Laborde Carmen , Mehring Cindy , Craig Teresa
Published: June 2021

We tested the hypothesis that estrogen (E) protects against high salt hypertension by attenuating the activation of the sympathetic nervous system (SNS). Dahl salt sensitive (SS) and salt resistant (SR) female rats were fed a 4% NaCl diet. Blood pressure (BP) was measured by radiotelemetry. DS and DR rats were divided into three groups: Intact, ovariectomized (OVX), and OVX with E replacement. Eight weeks after OVX or sham surgery, SNS activation was assessed by urinary excretion of norepinephrine (NE), and by the BP response to ganglionic blockage (GB). OVX DS rats had a significantly higher BP compared to Intact DS rats (175 ± 6 vs. 153 ± 5 mm Hg, respectively). E replacement after OVX prevented the augmentation of BP in DS rats (152 ± 6 mm Hg). OVX did not affect BP in DR rats. Urinary NE was significantly greater in OVX DS compared to Intact DS rats (40 ± 8 vs. 21 ± 2 ng/ml, respectively). E replacement prevented the increase in NE excretion after OVX in DS rats (20 ± 1 ng/ml). OVX did not affect NE excretion in DR rats. GB caused significant decreases in BP in all DS and DR rats, which were greater in DS compared to DR rats. In DS rats, the fall in BP with GB was augmented in OVX compared to Intact, which was prevented with E replacement. We conclude that estrogen loss augments, and estrogen replacement attenuates the contribution of the sympathetic nervous system to hypertension in female SS rats on high salt diet. Supported by NIH/NIA AG20256.