bluuebunny/crossref_metadata_2025 huggingface dataset:
Circulating oxidized low‐density lipoproteins are associated with overweight, obesity, and low serum carotenoids in older community‐dwelling women
Authors: Beck Justine C , Ferruci Luigi , Sun Kai , Linda Fried , Varadhan Ravi , Walston Jeremy , Guralnik Jack , Semba Richard
Published: June 2021

Context:

Oxidized low‐density lipoproteins (ox‐LDL) have been implicated in the pathogenesis of atherosclerosis and cardiovascular disease. The relationship between serum carotenoids, which are important components of low‐density lipoproteins, and obesity with ox‐LDL has not been well characterized.

Objective:

The objective of this study was to examine the independent relationships between total serum carotenoids and obesity with ox‐LDL in moderately to severely disabled older women living in the community.

Methods:

Serum carotenoids and oxLDL were measured in 543 women from the Women's Health and Aging Study I in Baltimore, Maryland.

Results:

Total serum carotenoids, smoking, overweight (BMI 25–29.9 kg/m2), and obesity (BMI ≥30 mg/kg2) were significantly associated with ox‐LDL/LDL‐cholesterol ratio after adjusting for age, C‐reactive protein, and chronic diseases. Alpha‐tocopherol and selenium were not significantly associated with ox‐LDL/LDL‐cholesterol ratio.

Conclusions:

Older women who are overweight or obese or who have low total serum carotenoids are more likely to have higher lipoprotein oxidation. Weight reduction in overweight/obese women and increased intake of carotenoid‐rich foods may potentially reduce lipoprotein oxidation.

This work was supported by National Institute on Aging Grant R01 AG027012, AG11703‐01A1, NIH‐NCRR, OPD‐GCRC grant RR00722, R01 AI41956, NIA Contract N01‐AG12112, and the Intramural Research Program, National Institute on Aging, NIH.
The role of CD36 and Caveolin‐1 in atherosclerotic lesion formation
Authors: Guerin Theresa , Thomas Candice , Smart Eric J
Published: June 2021

While CD36 has been found to have both positive and negative effects on the development of atherosclerosis, caveolin is thought to be cardioprotective. LDL receptor (LDLr) null mice form atherosclerotic lesions when fed a high fat (lard) diet. In this study, we examined the roles of CD36 and Caveolin‐1 (cav‐1) on the formation of atherosclerotic lesions in a LDLr null background. LDLr null, CD36/LDLr null, and Cav‐1/LDLr null mice were fed a high fat (lard) diet, myristic acid diet, linoleic acid diet, or a stearic acid diet for 9 – 13 weeks. At the end of the study, the mice were processed to quantify atherosclerotic lesions. On high fat (lard) diet, CD36/LDLr null mice had a dramatic increase in atherosclerotic lesions whereas the Cav‐1/LDLr null mice had a decrease compared to LDLr null mice. In contrast, on the myristic acid, linoleic acid, and stearic acid diets, the LDLr null mice produced significantly more lesions than both CD36/LDLr and Cav‐1/LDLr null mice. The stearic acid diet did not alter the extent of lesion formation between Cav‐1/LDLr and CD36/LDLr null mice however, the myristic acid and the linoleic acid diets increased lesion formation in the Cav‐1/LDLr null compared to the CD36/LDLr null mice. In conclusion, these data indicate that the role of CD36 in atherosclerosis is at least partially determined by the type of fat in the diets, whereas the absence of Cav‐1 seems to be protective.
AMPK and Akt crosstalk controls dura mater post ovariectomy (OVX) microvascular remodeling in pigs
Authors: Glinskii Olga V , Glinsky Vladislav , Abraha Tsghe , Huxley Virginia
Published: June 2021

Microvascular remodeling associated with the onset of reproductive senescence is a complex multi‐step process. Recently, we demonstrated that cessation of ovarian hormone production causes dramatic remodeling of meningeal microvascular networks, characterized by significant capillary rarefaction. However, the initial estrogen‐dependent post OVX loss of microvessels triggers stromal and vascular hypoxic responses leading to an increase in HIF‐1α expression and PDGF/VEGF system activation. Concomitant attenuation of hypoxia‐induced phosphorylation of AMP‐activated protein kinase (AMPK), caused by reduced estrogen (E2) levels, contributes to the activation of Akt and mTOR/p70S6K pathways, thereby promoting enhanced protein synthesis and prompting new vessel growth. Indeed, 2 months post OVX, we observed considerable angiogenic activity in meningeal microvascular networks that underwent previous regression. In summary our results demonstrate that post OVX angioadaptation proceeds through distinct stages regulated tightly on multiple levels by a complex interplay between several major signaling pathways induced by an array of internal and external metabolic stimuli. Supported by: NASA NNJ05HF37G, VA Merit Review Award, and NIH HL078816, HL075186, RR017353.
Disruption of angiopoietin‐1/Tie‐2 signaling contributes to the impairment of myocardial vasculature maturation and angiogenesis in db/db mice
Authors: chen Jian‐xiong , Stinnet Amanda , Zeng Heng
Published: June 2021

We hypothesized that disruption of angiopoietin‐1/Tie‐2 signaling in diabetes would result in impairment of smooth muscle recruitment and vasculature maturation, contributing to impaired myocardial angiogenesis. Our data revealed that myocardial Tie‐2 expression was significantly attenuated whereas angiopoietin‐2 (Ang‐2) was sustained expression in the db/db mice subjected to myocardial ischemia. Interestingly, angiopoietin‐1(Ang‐1)‐induced smooth muscle cells recruitment and the number of smooth muscle cells coverage per vessel area of the infarct vasculature were significantly reduced in db/db mice. This was accompanied by a markedly impairment of ischemia‐induced myocardial capillary and arteriole densities. Systemic administration of adenovirus Ang‐1 rescued the impaired myocardial angiogenesis and smooth muscle cells recruitments in db/db mice. Our data showed that scar formation was significantly reduced in Ad‐Ang‐1 treated db/db mice compared with Ad‐GFP db/db mice. Additionally, hyperglycemia significantly impaired Ang‐1‐induced angiogenic signaling, along with remarkably inhibition of Ang‐1‐induced angiogenic responses both in vitro and ex vivo. Overexpression of Ang‐1 significantly improved the impaired angiogenic responses under hyperglycemic conditions. Our findings underscore the important role of angiopoietin‐1/Tie‐2 signaling in diabetes‐induced impairment of angiogenesis.
Tissue Kallikrein increases perivascular cell coverage of angiogenic vessels via a Bradykinin‐2‐Receptor dependent mechanism
Authors: Stone Oliver Andrew , Emanueli Costanza , Harper Steven J. , Madeddu Paolo , Bates David O.
Published: June 2021

We previously showed that Tissue Kallikrein (hTK) overexpression induces an increase in perivascular cell coverage of angiogenic vessels. To further investigate the mechanisms of hTK induced angiogenesis, hTK was overexpressed in the adult rat mesentery, and intravital and confocal imaging used to examine vessel morphology and histology following daily subcutaneous injection of the B2‐receptor antagonist Icatibant. B2R blockade caused no significant reduction in the hTK induced increase in functional vessel area (457±90% vs 420±74%). Angiogenesis in the presence of Icatibant was associated with a significant reduction in fractional pericyte coverage (64±4 vs 27±6 %, p<0.02) and smooth muscle cell recruitment was abolished (7.2±1.2% vs 0±0% p<0.001). Icatibant also caused an increase in sprout point density (7±2 vs 22±3mm−2, p<0.05) and was associated with haemorrhage of the microvasculature. Further investigation indicated the co‐localisation of pericytes and B2R expression in both control and treated animals. These data provide the first mechanistic explanation for the hTK induced increase in pericyte coverage, implicating a pivotal role for the B2 receptor in the recruitment/proliferation of perivascular cells and the stabilisation of the angiogenic microvasculature.

All data presented mean±SEM, n = 6. Work funded by the BHF.
Angiopoietin‐1 modifies multiple microvessel permeability coefficients in non‐inflamed vessels
Authors: Salmon Andrew , Harper Steven , Bates David
Published: June 2021

Angiopoietin‐1 (Ang1) is widely expressed by pericytes and induces constitutive phosphorylation of the endothelial cell receptor Tie2. Ang1 reduces neutrophil adhesion and solute flux following inflammatory stimulation. We examined the hitherto untested hypothesis that Ang1 modifies permeability coefficients in non‐inflamed microvessels. Continuous microvessel hydraulic conductivity (LP: x107 cm.s−1.cmH2O−1) and reflection coefficient (σ) were measured in frog mesenteric microvessels with the Landis‐Michel assay. Ang1 (200 ng.ml−1) caused a significant rise in σ (vehicle: 0.78±0.03; Ang1: 0.91±0.03; p<0.05, paired t‐test (n=10)) and a significant reduction in LP (vehicle: 2.0±0.9; Ang1: 1.2±0.9; p<0.05, Wilcoxon (n=11)). Fenestrated microvessel LP‐area product (LPA: nl.min−1.mmHg−1), measured in isolated rat glomeruli with an oncometric assay, also reduced significantly in response to Ang1 (vehicle: 1.0±0.1 (n=31); Ang1: 0.8±0.1(n=30); p<0.05, unpaired t‐test). Ang1 may induce a plethora of changes in different microvessels that alter resistance to water and albumin movement, or may modify a determinant of microvascular permeability coefficients that is common to both continuous and fenestrated microvessels.
Differing Effects of Cilostazol and Aspirin on Thrombus Formation and Microvascular Permeability
Authors: Kajimura Mayumi N.A. , Morikawa Takayuki N.A. , Suematsu Makoto
Published: June 2021

Cilostazol is a unique antiplatelet drug as it has been shown to reduce the recurrence of vascular events without exacerbating a risk of bleeding complication, although mechanisms for such effects are unknown. We examined its effects on changes in formation of platelet thrombus and compared those with aspirin. Microhemorrhage was induced by laser ablation on a rat mesenteric venule to observe a spatiotemporal process of non‐occlusive thrombus formation. Platelet thrombi in both cilostazol‐ and aspirin‐treated rats were examined to choose the doses of reagents resulting in comparable anti‐platelet effects. Surprisingly, cilostazol, but not aspirin, was found to reduce erythrocyte extravasation under these conditions. To test if such an effect is attributed to modulation of endothelial barrier properties, we measured changes in water permeability (Lp) and reflection coefficient to albumin (σ) in single‐perfused venules. Cilostazol significantly abolished a transient increase in Lp and a decrease in σ when vessels were challenged by histamine. Furthermore the reagent attenuated baseline Lp under a control state. These results indicate that cilostazol not only acts on the platelet to reduce the size of developing thrombus, but also acts on endothelium to tighten up the endothelial barrier at least in part by inhibiting the cAMP‐degrading enzyme in the endothelium that differs from aspirin. Supported by MEXT.
Nicotine‐induced up‐regulation of Wingless/Int signaling in human alveolar interstitial fibroblasts (AIFs)
Authors: Cerny Laura , Sakurai Reiko , Wang Ying , Guo Pinzheng , Torday John S , Rehan Virender K
Published: June 2021

Nicotine induces AIF‐to‐myofibroblast (MYF) transdifferentiation. Since these phenotypes are determined by Peroxisome Proliferator‐Activated Receptor (PPAR)γ and Wnt signaling, respectively, we hypothesized that nicotine down‐regulates PPARγ signaling and up‐regulates Wnt signaling. And since nicotine activates PKC, we hypothesized nicotine up‐regulates Wnt signaling via PKC activation. WI38 cells were treated with nicotine 10−6M for 30 min or 24 hours +/− 30 minute pretreatment with calphostin C (10−7), a pan‐PKC inhibitor. Cell lysates were analyzed by Western blotting for activation of 1) PKC (p‐PKC) and 2) Wnt signaling (p‐GSK‐3β, β‐catenin, LEF‐1, and fibronectin); and 3) down‐regulation of PPARγ. We also examined activation of nicotinic acetylcholine receptors (nAChR)‐α3 and –α7, up‐stream regulators of Wnt signaling.

Following 30 min of nicotine stimulation, nAChR‐α3 and –α7 were up‐regulated, accompanied by activation of PKC and Wnt signaling and significant changes in expression of down‐stream targets of Wnt signaling at 24h. All changes were blocked by calphostin C, indicating the central involvement of PKC in nicotine‐induced activation of Wnt signaling, and hence AIF‐to‐MYF transdifferentiation. Therefore PKC signaling plays a central role in nicotine‐induced activation of Wnt signaling, providing novel preventive/therapeutic targets. Funding‐NIH HL75405 & TRDRP 14RT‐0013.
exoY increases Pseudomonas aeruginosa virulence
Authors: Prasain Nutan , Alvarez Diego F. , Frank Dara W. , Stevens Troy
Published: June 2021

Pseudomonas aeruginosa is a principal cause of pneumonia that progresses to sepsis and acute lung injury. Bacterial virulence is due to the type III secretion system (TTSS), which is a functional bacterial needle that introduces exotoxins into host cells. Four known exotoxins are introduced through the TTSS; exoY is a cytosolic adenylyl cyclase that disrupts the endothelial barrier and causes edema. It is not clear whether exoY contributes to P. aeruginosa virulence. Survival analysis was performed in rats exposed to three P. aeruginosa strains, including strains that possess a: (1) functional TTSS, but only inject exoY (exoY+), (2) functional TTSS, but inject a catalytically inactive exoY (exoYK81M), and (3) non‐functional TTSS (exoYTM). Bacteria were introduced by tracheal installation, and survival curves generated over a 7‐day time course. Virulence was greatest in animals treated with exoY+ (LD50 ≈ 108 CFU); exoYK81Mor exoYTM strains were not virulent (LD50 > 109 CFU). Histological inspection of exoY+‐treated animals revealed large areas of patchy atelectasis, with significant neutrophil infiltration into alveoli. Alveolar fluid accumulation was prominent, and large peri‐vascular cuffs were observed, consistent with increased endothelial permeability. Thus, introduction of catalytically active exoY by P. aeruginosa is an important determinant of pathogenic virulence. HL‐60024 & HL‐66299
Angiopoietin‐1 reverses protein permeability in an injury model of cultured human alveolar epithelial type II cells
Authors: Neyrinck Arne Pieter , Lee Jae‐Woo , Fang Xiaohui , Gupta Naveen , Matthay Michael A
Published: June 2021

OBJECTIVE: We recently reported that acute lung injury (ALI) pulmonary edema and cytomix (IL1β, TNFα and IFNγ) increase epithelial protein permeability (JBC, 2007). Angiopoietin‐1 (Ang‐1) maintains vascular integrity and reduces endothelial activation in a model of ALI (AJRCCM 2007). In this study, we hypothesized that Ang‐1 could reverse increased protein permeability in cultured human alveolar epithelial type II cells (ATII) exposed to cytomix.

METHODS: Isolated human ATII cells were cultured in an air‐liquid interface and protein permeability was measured. Cells were exposed to control medium, cytomix (50 ng/ml) and cytomix with recombinant human Ang‐1 (10,000 pg/ml).

RESULTS: 1) Cytomix increased protein permeability in cultured ATII cells. 2) Recombinant human Ang‐1 decreased ATII protein permeability significantly after exposure to cytomix (Figure 1).

CONCLUSIONS: Ang‐1 can reverse protein permeability across ATII cells exposed to cytomix. These results are the first results to show a potential role for Ang‐1 in regulation of alveolar epithelial permeability. The data support a further role of Ang‐1 in the treatment of ALI.

Supported by NIH HL51856 and HL51854
Systemic osmotic signaling pathways function upstream of WNK and GCK‐VI kinases to regulate hypertonic stress resistance in C. elegans
Authors: Choe Keith Patrick , Strange Kevin
Published: June 2021

The GCK‐VI subfamily of Ste20 kinases regulate volume‐sensitive ion channels and transporters. With no lysine (K) or WNK kinases interact with and activate the mammalian GCK‐VI kinases, PASK and OSR‐1. We recently reported that the C. elegans GCK‐VI kinase, GCK‐3, interacts and functions with the WNK kinase, WNK‐1, to regulate systemic volume and survival during hypertonic stress. The pathways that function upstream of WNK and GCK‐VI kinases are not defined. In C. elegans, cuticle collagens (e.g., DPY‐10) and novel proteins that are secreted from an epithelium near the cuticle (e.g., OSM‐11) have been postulated to be components of systemic mechanosensors that detect hypertonic shrinkage. Mutations in DPY or OSM proteins cause an osmotic stress resistant phenotype (Osr) characterized by elevated levels of the organic osmolyte glycerol and survival during exposure to hypertonic stress that is lethal to wild type worms. To determine if WNK‐1 or GCK‐3 function downstream from DPY or OSM proteins, we performed RNAi experiments. Knockdown of either kinase suppressed Osr in DPY and OSM mutants, but did not suppress glycerol accumulation or alter systemic volume. Our results demonstrate that WNK‐1 and GCK‐3 function downstream from systemic osmotic sensors to regulate hypertonic stress resistance by mechanisms that are independent from glycerol accumulation and volume regulation.
CFTR anion channel regulation by phosphorylation of focal adhesion kinase at tyrosine 407 in osmosensing mitochondria‐rich ion transporting cells of killifish (Fundulus heteroclitus)
Authors: Marshall William S , Katoh Fumi , Watters Kaitlyn D , Goudie Megan T , Cozzi Regina R F
Published: June 2021

Focal adhesion kinase (FAK) is a tyrosine kinase with multiple Y sites and an ezrin/radixin/moesin binding site. Antibodies to the phosphorylated form of FAK at Y407 and Y587 are colocalized with the Cl− channel CFTR in the apical membrane of mitochondria‐rich (MR) cells of seawater‐acclimated euryhaline killifish opercular epithelium. Hypotonic treatment (80%, 224 mOsm/kg) causes rapid, reversible cell swelling (observed via confocal microscopy of living cells in situ), inhibition of Cl− secretion and dephosphorylation of FAK pY407 but no effect on FAK phosphorylated at Y576. Re‐phosphorylation of FAK at Y407 specifically in the apical membrane of MR cells was immunohistochemically detected following 10‐min treatments with forskolin (10 μM), 3‐isobutyl‐1‐methylxanthine (IBMX, 0.1 mM) and isoproterenol (1.0 μM), all known to increase cAMP and activate CFTR. There is parallel recovery of Cl− secretion by MR cells in the isolated epithelium, detected electrophysiologically. These results suggest involvement of phosphorylation of FAK pY407 but not pY576 in cell signaling pathways regulating the Cl− transport function of CFTR. (Supported by NSERC Canada)
Phosphorylation of Serine‐256 is essential for downstream polyphosphorylation of AQP2
Authors: Møller Hanne B , Hoffert Jason D , Rützler Michael , Praetorius Jeppe , Knepper Mark A , Fenton Robert A
Published: June 2021

Phosphorylation of AQP2 at S256 is essential for its membrane targeting. Recently, we determined that S261, S264 and S269 residues are also phosphorylated by AVP and have begun to examine their role in AQP2 trafficking. MDCK cells stably expressing constitutively non‐phosphorylated forms of AQP2 demonstrated that single mutations (S261A, S264A and S269A) had no significant effect on apical membrane targeting following either AVP or forskolin treatment. A mutation mimicking constitutively phosphorylated AQP2 at position S269 (S269D) resulted in predominantly apical membrane localization of AQP2 even in the absence of stimulation. Osmotically induced swelling assays determined that rates of swelling in S269D‐AQP2 mutants were increased relative to wt‐AQP2 or S269A‐AQP2. Time course studies determined that S256‐AQP2 phosphorylation occurs first, raising the possibility that downstream phosphorylation is dependent on S256 phosphorylation. MDCK cells stably transfected with S256A‐AQP2 showed an elimination of S264 or S269 phosphorylation, whereas WT‐AQP2 or S256D‐AQP2 transfected cells exbihited a high level of phosphorylation at these sites. Thus, these data are compatible with the view that S256 phosphorylation is required for downstream phosphorylation.
Role of Epac in cAMP mediated PKA independent mechanism of intestinal Cl‐secretion
Authors: Kazi Mirajul Hoque , Van Rossum Damian B , Nick Zachos C , Chen Linxi , Guggino Sandra E , Tse Chung‐Ming
Published: June 2021

cAMP is a major regulator of Cl−secretion. With the recognition that the exchange protein directly activated by cAMP (Epac) transduces cAMP signaling cascades independent of protein kinase A (PKA), we test the hypothesis that both PKA and Epac are involved in forskolin (FSK) stimulated Cl− secretion. FSK is an adenylate cyclase activator. T84 cells were used for short circuit current (Isc) measurement in the Ussing chamber. FSK stimulated Isc was completely inhibited by the additive effects of the PKA inhibitor, H89 (1μM) and the [Ca2+]i chelator, BAPTA‐AM (25μM), suggesting the presence of both PKA dependent and independent mechanisms. Epac1 was found to be expressed in T84 cells. Both FSK and the Epac selective agonist, 8‐pCPT‐2‘‐O‐Me‐cAMP (8‐CPT, 50μM), elevated [Ca2+]i. Activation of Epac1 by 8‐CPT induced Isc in intact T84 cells and in cells permeabilized with nystatin at the basolateral membrane. This effect of 8‐CPT was completely abolished by BAPTA‐AM, but not by H89 (1μM) and the CFTR specific blocker, cftr‐172, suggesting that 8‐CPT stimulated non‐CFTR channel(s) via elevation of [Ca2+]i. FSK stimulated Isc was inhibited by the PLC inhibitor U73122. Both FSK and 8‐CPT increased the amount of activated (GTP‐bound) Rap proteins, which have been shown to activate PLCε to mobilize [Ca2+]i. Our findings in T84 cells were further validated in mouse ileum. 8‐CPT stimulated Isc in mouse ileal sheet and this stimulation was not inhibited by H89. We conclude that PKA and Epac1 contribute to FSK stimulated Cl− secretion in intestine via PKA dependent and Ca2+ dependent pathways, the latter mediated by Epac1‐Rap2‐PLC‐Ca2+ signaling involving non‐CFTR channel(s).
Elucidating Conformational Changes in the γ‐Aminobutyric Acid (GABA)Transporter
Authors: Meinild Anne‐Kristine , Loo Donald , MacAulay Nanna
Published: June 2021

In addition to the Na+‐coupled GABA transport, the Na+/Cl− dependent GABA transporter, GAT‐1, also has a Li+‐induced leak. Recently, the amino acids directly involved in the interaction with the Na+ and Li+ ions have been identified (Zhou et al, 2006, JBC Vol. 281). In the present study, we obtain insight into the structural dynamics of the two transport modes of GAT‐1 by applying the voltage clamp fluorometry method (VCF) to wild type GAT‐1 expressed in Xenopus oocytes. We have taken advantage of the only accessible cysteine in wild type rat GAT‐1, C74, and labeled this residue with a cysteine‐reactive fluorophor, tetramethyl rhodamine‐6‐maleinamide.

Our fluorescent read‐outs show that the transporter undergoes different voltage dependent conformational changes in the presence of the different ions in the external solution: in Na+ buffer the V½ for fluorescence is −2 ± 9 mV whereas in Li+ buffer it is −71 ± 12 mV. The time courses of these fluorescent changes are also highly dependent on the cation: in Na+ the τmax is 120 ms and in Li+ it is 40 ms. The fluorescence data for Li+ correlates with the Li+ induced currents: these are highly voltage dependent and only present at hyperpolarized potentials (<−50 mV).

The study demonstrates the advantage of applying the VCF method in combination with homology modeling of the neurotransmitter transporters to the newly crystallized bacterial homolog, LeuT.
Novel Ouabain Receptors in Bovine Adrenal Cortex: Evidence for Ouabain Transport
Authors: Liu Yong , Ward Stephen C. , Hamilton Bruce P. , Hamlyn John M.
Published: June 2021

Novel high affinity binding sites for ouabain (NOR) are present in bovine adrenal cortex (Ward SC, Hypertension 2002; 39[pt. 2]:536). To distinguish NOR from classical sodium‐potassium pumps (Na pumps), we used fresh adrenal cortical membranes. Scatchard analysis showed that K+ (20mM) reduced the apparent Kd for ouabain binding to Na pumps from 4.6 ± 0.2 to 240 ± 7.2 nM with no change in Bmax (10.1 ± 0.1 vs. 10.3 ± 0.2 pmoles/mg). K+ increased the Kd for ouabain binding to NOR and increased the Bmax (0.15 ± 0.02 vs. 0.26 ± 0.06 pmoles/mg). Moreover, the effect of Na+ was more dramatic; Na+ inhibited ouabain binding to Na pumps with no change in Bmax, while Na+ increased the apparent Kd for ouabain binding to NOR and increased Bmax > 25‐fold. Ouabain binding to the Na pump in stored membranes remained constant for up to 50 days whereas NOR related binding declined with a t1/2 of 6.8 days. The association of ouabain with membranes was 0.538 pmoles/mg when [Na+]o » [Na+]i, and was 0.152 pmoles/mg when [Na+]o « [Na+]i, and was sensitive to permeable anions. NOR binding was sensitive to osmolarity. Photoactive analogs of ouabain labeled a 35 kDa polypeptide. We conclude that NOR mediates the secondary active sodium‐dependent transport of 3H‐ouabain into sealed membrane vesicles and that this process is electrogenic. NOR may mediate the secretion of cardiac glycosides by the adrenal cortex in vivo. Supported by HL078870 and HL075584.
PKCδ‐SPAK (Ste20‐related proline alanine‐rich kinase) interaction in the regulation of native human NKCC1
Authors: Liedtke Carole M. , Smith Laura
Published: June 2021

Activation of native NKCC1 by hyperosmotic stress involves a regulatory proteome which includes PKCδ interacting directly with the actin cytoskeleton and with SPAK kinase. NKCC1 activation requires phosphorylation, yet PKCδ does not directly phosphorylate NKCC1. The goal of this study is to determine whether SPAK is part of a kinase cascade linking PKCδ to phosphorylation of native human epithelial NKCC1. NKCC1 activity was measured as bumetanide‐sensitive basolateral to apical 86‐Rb flux in Calu‐3 cells. SPAK was downregulated using siRNA delivered into Calu‐3 cells using an Amaxa nucleofector. siSPAK reduced SPAK protein expression by 73.5% without altering expression of NKCC1, PKCδ, and actin and prevented activation of NKCC1 by hyperosmotic stress. Substitution of a non‐targeting siCONTROL RNA did not affect expression of SPAK, PKCδ, NKCC1, and actin or activation of NKCC1. Using recombinant proteins, we demonstrate direct binding of PKCδ to SPAK and PKCδ‐mediated activation of SPAK. In intact cells, hyperosmotic stress increased native phosphorylated PKCδ (pPKCδ) 2.4‐fold, indicating activation of PKCδ, the amount of PKCδ and pPKCδ coimmunoprecipitating with SPAK 2.9‐fold and 3.6‐fold, respectively, and the amount of SPAK coimmunoprecipitating with PKCδ by 1.9‐fold. The results indicate that PKCδ mediates the stress signal to SPAK. Supported by a grant from the NIH, RO1‐HL‐58598.
Human Plasma Membrane Anion Exchanger, AE1: Over‐expression in Saccharomyces cerevisiae
Authors: Bonar Pamela Therese , Casey Joseph
Published: June 2021

Anion exchanger 1 (AE1) facilitates the exchange of chloride for bicarbonate across the plasma membrane of renal and erythrocyte membranes. The 55 kDa membranedomain of AE1 alone is responsible for its transport function, however a high resolution structure of this domain has not been solved. The limiting factor to obtaining acrystal structure is the availability of homogenous, purified AE1. Here we developed a novel expression and efficient purification system for AE1, using S. cerevisiaeto allow for large‐scale expression, while maintaining proper folding of AE1. The expression construct consists of the AE1 membrane domain (AE1MD), residues 388–911 of human AE1, followed by the 9 C‐terminal amino acids of rhodopsin. Expression was under the control of the constitutive promoter and transcriptionalterminator of the yeast H+‐ATPase gene. AE1MD was expressed in S. cerevisiae, strain BJ5457, to 0.3 mg AE1MD per litre of culture. We characterized the cellularlocalization of AE1MD by confocal immunofluorescence microscopy and sucrose gradient ultracentrifugation. AE1MD structure was assessed by binding to animmobilized anion exchange inhibitor (SITS‐Affi‐gel). Once solubilized by the detergent n‐dodecyl‐β‐D‐maltoside, AE1MD was purified with a 1D4 antibody affinitycolumn, which recognizes the rhodopsin tag on AE1MD. This research is supported by the Canadian Institutes of Health Research.
hIK1 potassium channel in the G1 phase of the cell cycle (Preliminary Report)
Authors: Chao Pin‐Chun , Hamilton Kirk L.
Published: June 2021

Potassium (K+) channels are important in cell growth, proliferation and the progression of cells through phases of cell cycle. The human intermediate‐conductance Ca2+‐activated K+ channel (hIK1) has been suggested to be crucial for progression of cells through the cell cycle, as inhibiting the channel can reduce cell proliferation. It is hypothesized that the cellular expression of hIK1 changes as the cell progresses from early G1 phase to G1/S boundary in LNCaP cells, a human metastatic prostate cancer cell line. In order to study the expression pattern of hIK1 at different phases of the cell cycle, the first step was to develop a protocol for arresting LNCaP cells. Fluorescence activated cell sorting (FACS) was used in this project. Our results demonstrated, for the first time, that LNCaP cells were systematically arrested by serum starvation at different phases of the cell cycle. These data suggested that the cells could be arrested at G1 (~80%), late G1 (~83%), G1/S boundary (~65%/~28%), and shifted into the G2/M phase after the addition of thymidine for 24 hours followed by incubation in normal medium for 9 hours. We have also confirmed the specificity of an antibody for examining the cellular/surface expression of native hIK1 as LNCaP cells progress thought the cell cycle. The work will provide a basic understanding of the role of hIK1 in cell proliferation of prostate cancer cells.
Complex Regulation of Ca2+‐Activated Cl− Channels by Phosphatases in Pulmonary Arterial Smooth Muscle Cells
Authors: Ayon Ramon J , Sones William R , Forrest Abigail , Perrino Brian , Greenwood Iain A , Leblanc Normand
Published: June 2021

Ca2+‐activated Cl− currents (ICl(Ca)) represent a major excitatory mechanism in vascular smooth muscle cells (SMCs). Our group previously showed that CaMKII and Calcineurin (CaN) exert opposed regulatory effects on ICl(Ca) in arterial SMCs. The specific aim of this study was to determine whether other phosphatases modulate ICl(Ca) in rabbit pulmonary artery (PA) myocytes. RT‐PCR performed on rabbit brain, lung and PA cDNA revealed single bands of the expected product size for PP1, PP2A and CaN in all tissues studied. In a previous study, our group (Ayon et al., Faseb J. 21: 873.5, 2007) showed NIPP‐1, a peptide inhibitor of PP1, blocked recovery of ICl(Ca) in cells dialyzed with no ATP. In contrast, Fostriecin, a specific inhibitor of PP2A, had no effect on ICl(Ca) recovery. To further test the PP1 hypothesis, we examined the effects the catalytic subunits of PP1 and PP2A have on ICl(Ca) in cells dialyzed with 3 mM ATP. Surprisingly, PP1 failed to offset ICl(Ca) rundown, while filling the intracellular milieu with PP2A led to ICl(Ca) recovery. The results of our experiments revealed differential responses when manipulating endogenous phosphatases as opposed to supplying these phosphatases exogenously. These results highlight unsuspected complex mechanisms of regulation of ICl(Ca) by phosphatases in PASMCs. [NIH (HL075477 and NCRR 5P20 RR15581), British Heart Foundation (PG/05/038), The Wellcome Trust]
Further characteristics of hyposmotically activated K loss in human lens epithelial cells (hLECs)
Authors: Lauf Peter K. , Chimote Ameet A. , Adragna Norma C.
Published: June 2021

Regulatory volume decrease of hLECs in hyposmotic (150 mOsM) media involves KCa3.1 (IK) channel activation and cell water loss. High external K and clotrimazole (CTZ), but not a variety of anion channel blockers, were inhibitory. Extending these findings, we studied the selective IK channel blocker (TRAM‐34 [1‐[(2‐chlorophenyl) diphenylmethyl]‐1H‐pyrazole]), and additional anion channel blockers used in other systems, especially DIOA {[(dihydro‐indenyl)oxy]alkanoic acid]}, phloretin and DCPIB [4‐2(butyl‐6,7‐dichloro‐2‐cyclopentylindan‐1‐on‐5‐yl)oxybutyric acid]. Ouabain‐ and bumetanide‐insensitive K loss and Rb influx were measured in hLECs (FHL124) by atomic absorption spectrophometry. TRAM‐34 (>50 μM) reduced K loss by ~ 30% with little effects on Rb influx, an asymmetry contrasting with the total inhibition of both fluxes by CTZ, also an inhibitor of P450, suggesting at least two different mechanisms either at the K channel and/or regulatory levels. Phloretin (0.25–1 mM) reduced Rb influx by ~60%, less affecting K loss. In Cl or NO3, 300 μM DIOA effectively inhibited K loss, but not Rb influx, a concentration tenfold higher than used to block K‐Cl cotransport. DCPIB (50 μM) mainly reduced K‐loss. Hence, hyposmotic K loss, through putative IK channels in hLECs, is associated with and/or rate‐limited by Cl loss through DIOA‐and phloretin‐sensitive anion channels whose nature awaits elucidation.
Detection of extracellular ion gradients enables characterization and functional mapping of ion channels and transporters
Authors: Messerli Mark A. , Kurtz Ira , Smith Peter J.S.
Published: June 2021

Conventional, voltage clamp methods have not been successful for noninvasive characterization of ion channels, studying the vast majority of slow rate and no‐current passing (electroneutral) transporters, or the direct identification of transport stoichiometry. It is for these reasons that we explored the use of electrochemical sensors for near real time measurement of extracellular ion gradients that result from ion passage through channels and transporters. Ion‐selective electrodes possess the speed and sensitivity to capture extracellular ion activity changes from single ion channels. We monitored Ca2+‐activated K+ channel activity expressed in CHO cells using a fast response K+‐selective electrode. The K+ gradients measured using this approach fit well with a diffusion‐based model, enabling noninvasive, functional characterization of the channels and mapping of their location. When ion‐selective electrodes were used in a modulation mode, termed self‐referencing, the signal to noise ratio was increased, enabling the measurement of small, relatively steady fluxes from ion transporters. Near real‐time characterization of slow rate and electroneutral transporters could be performed. In conclusion, we have developed a novel extracellular, self‐referencing microelectrode system that will be useful for the noninvasive, long‐term monitoring of ion transport in cells and epithelia. Supported by the NIH.
Different cell surface structures have different levels of ezrin phosphorylation
Authors: Zhu Lixin , Hatakeyama Jason , Shastri Aditi , Chen Cheng , Poon Kevin , Forte John G
Published: June 2021

Ezrin, a member of the ERM family, is a membrane‐cytoskeleton linker protein activated by phosphorylation on Thr 567 (T567P). In gastric parietal cells, high turnover of T567P on ezrin is necessary for membrane recycling and acid secretion. Studies using transformed cell lines have reported conflicting results regarding localization and translocation of ezrin with changing T567P levels, challenging the T567P turnover hypothesis. Here we examined this question in several tissues in situ where ezrin plays a pivotal functional role. Surprisingly, T567P levels in kidney and small intestine were >10 times higher than gastric glands. Blocking phosphorylation or dephosphorylation revealed rapid turnover of T567P in all 3 tissues. Immunofluorescence staining showed that decreased T567P did not dislocate ezrin from the respective apical membranes; rather, dephosphorylation of T567P only decreased binding of ezrin to F‐actin, but ezrin‐membrane interaction was unaffected, as indicated by the triton and digitonin extraction analyses. Thus, steady state T567P levels markedly differ at different cell surface structures, but they all show turnover activity consistent with the membrane‐cytoskeleton linker role of ERMs. The data are consistent with an hypothesis that surface plasticity depends on turnover of ERM bonding interactions to take up and relax tension between the plasma membrane and cytoskeleton.
Involvement of P2Y Receptors in TGFβ‐Induced EMT of MDCK Cells
Authors: Wolff Christine Irene , Bundey Richard A. , Insel Paul A.
Published: June 2021

Epithelial‐to‐mesenchymal transition (EMT) contributes to renal tubulointerstitial fibrosis, a common pathway leading to end‐stage renal disease. EMT can be induced in certain renal cells by transforming growth factor beta (TGFβ) and results in the phenotypic conversion of tubular epithelia to fibroblasts, reduction of E‐Cadherin expression and induction of α‐smooth muscle actin (αSMA). Since nucleotides can be released during renal injury, we hypothesized that P2Y (nucleotide) receptors contribute to EMT of renal epithelial cells. To test this hypothesis, we used a well‐characterized renal epithelial cell line, Madin Darby Kidney Cells (MDCK), and examined the role of P2Y receptors in TGFβ‐induced EMT. Quantative PCR experiments (used to identify P2Y receptor expression) revealed P2Y2 > P2Y11 > P2Y1 = P2Y14 > P2Y6 = P2Y13 > P2Y12 = P2Y4 as the relative order of abundance. TGFβ treatment reduced the expression of P2Y13 mRNA, elevated P2Y11 and P2Y14 mRNA levels without substantial changes in mRNA expression of P2Y1 and P2Y2. The P2Y agonist ATPγS inhibited protein expression of the EMT marker αSMA in response to TGFβ without influencing the E‐Cadherin decrease or morphological changes. These results indicate that TGFβ‐promoted EMT differentially regulates expression of P2Y receptor subtypes of MDCK cells and that P2Y receptor activation potentially blunts features of the EMT phenotype. Supported by grants from NIH.
GABA‐mediated inhibitory effects of nitric oxide in the gracile nucleus on sensory neuropathies in Zucker Diabetic Fatty rats
Authors: Rong Peijing , Ma Shengxing
Published: June 2021

The purpose of this work is to determine the effects of nitric oxide (NO) and GABA transmission in the gracile nucleus (GN) on functional neuropathic changes in Zucker Diabetic Fatty (ZDF) rats. A microdialysis probe was implanted into the GN and an infusion fluid was pumped at 3.0 μ/min. 3‐Morpholinyl‐sydnoneimine hydrochloride (SIN‐1, 10 μM) was dissolved in artificial cerebrospinal fluid (ACSF) and infused into the GN in the presence or absence of picrotoxin (30 μM). Time‐response curves were determined by measurements of the mechanical threshold and withdrawal latencies to hot and cold stimuli during infusion for 20 minutes and at 20, 40, and 60 minutes after microinfusion of the compounds. The withdrawal latencies to mechanical, cold and hot stimuli were significantly increased following infusion of SIN‐1 but not altered by infusion of ACSF. The responses to SIN‐1 on withdrawal latencies to mechanical, cold and hot stimuli were consistently inhibited by picrotoxin in the dialysis fluid. These results demonstrate that the functional neuropathic changes in ZDF rats are improved by administration of NO donor in the GN and the responses are blocked by inhibition of GABA transmission. We conclude that GABA transmission mediates NO effects in the GN, which produces an inhibitory modulation of somatosensory/nociceptive susceptibility in Type II diabetes. Supported by ADA 7‐07‐RA‐100 and NIH RO1 AT 002478 to S. Ma.
High‐throughout Screening of Circadian Rest/Activity Cycle Can Detect Significant Differences Estimates of Sleep/Wake Cycle In ENU Knockout Inbred Strains Of Rats
Authors: Feroah Thom R , Vernon Heather J , McQuestion Greg , Forster H V , Merritt A , Dwinell M , Moreno‐Quinn C , Greene A , Cowley A , Jacob H J
Published: June 2021

17 ENU‐mutant rat strains of both genders were screened via a new high throughput system for circadian rest/activity behavior. The ENU targeted genes were selected for their involvement in cardiovascular disease models. The screening system consisted of 12 plethysmographic chambers with movement acquired by horizontal and vertical sensors while the rats were exposed to 3 – 12 hr light:dark cycles (0800:2000 hrs). Based on previous analysis, movement during the last 24 hours in 5 strains (both genders; FHHMCW, FHH‐Lcat, FHH‐Slc8a2, FHH‐Ghsr, FHH‐Agtr1b) were analyzed for Awake (Aw), Quiet (Qt), Sleep (Sp) and Sustained Sleep (SS) periods and summarized per hour. All strains of both genders showed significant* circadian variation in Aw, Sp and SS except for Sp in FHH‐Slc8a2 males. FHH‐Slc8a2 males showed significant* decreases in SS (15–20%) during the light cycle and increases (9–13%) during dark cycle with significant* reciprocal changes in Aw compared to FHH males. These data show that high throughput screening can discriminate between different estimates of circadian sleep/wake cycle in ENU‐mutant rat strains. Furthermore, these data show that ENU targeted genes selected for one disease model may have effects on multiple systems that can be used for further genetic dissection of complex traits in rodent models susceptible to cardiovascular and sleep dysfunction. Study funded by NHLBI (U01 HL66579) (*P < 0.05)
Effects of partially‐hydrogenated fish oil on lipid and lipoprotein metabolism in guinea pigs
Authors: Kraft Jana , Michel Melanie , Plonne Dietmar , Almendingen Kari , Jahreis Gerhard
Published: June 2021

This study was designed to examine the effects of partially hydrogenated fish oil (PHFO), compared with fish oil (FO) used as a control, on serum lipids, lipoprotein profile and aortic lesion development in guinea pigs fed an atherogenic diet. Adult male Hartley guinea pigs (n=20; 10 per group) were fed a diet supplemented with 8.5 % PHFO or FO for 12 weeks. Diets contained 0.25 % cholesterol (C). The total trans fatty acid content of the PHFO diet was 4 %. PHFO‐fed animals had a significantly increased serum concentrations of triacylglycerol, total cholesterol, LDL‐C, and sdLDL‐C (P<0.001) compared with the FO group. In contrast, serum HDL‐C concentrations did not differ between the groups. Analysis of the ascending aorta and aortic arch revealed no lesion development in both treatment groups but PHFO‐fed guinea pigs showed a distinct lipid accumulation in the aortic arch when compared to the FO group. Results from this study demonstrate that guinea pigs provide a good model for studying the effects of PHFO on the lipid/lipoprotein metabolism. However, no signs of atherosclerosis in the aorta (lesion development) could be established under the used experimental conditions.
Oxidative Stress and Angiotensin Receptor Expression in Heart Failure
Authors: Liu Dongmei , Gao Lie , Talbitzer Kaye , Zucker Irving H.
Published: June 2021

Angiotensin type I receptors (AT1R) and Angiotensin type II receptors (AT2R) have been shown to counteract each other in physiological and pathological states. However, it is not clear if ROS regulates Ang II receptor protein. The present study investigated if oxidants can regulate the expression of angiotensin receptors in the RVLM of CHF rabbits, and their relationship to the JNK/MAPK pathway and to the transcription factor activator protein 1(AP1). CHF rabbits were randomly divided into two groups. One group (n=5) was treated with intracerebroventricular (ICV) Tempol, the other group was treated with ICV vehicle (n=5). Compared to vehicle treated CHF rabbits Tempol significantly decreased AT1R mRNA expression (1.0 ± 0.16 vs. 0.54 ± 0.05, P<0.05) and AT1R protein expression (1.6 ± 0.29 vs. 0.88 ± 0.16, P<0.05); phosphorylated Jnk protein (0.10 ± 0.02 vs. 0.31 ± 0.10, P<0.05); and phosphorylated c‐Jun (0.14 ± 0.05 vs. 0.02 ± 0.001, P<0.05). Interestingly, compared to the vehicle treated group Tempol up regulated AT2R mRNA expression (1.0 ± 0.1 vs. 1.7 ± 0.16, P<0.01) and protein expression (0.11 ± 0.06 vs. 0.63 ± 0.20, P<0.05) in RVLM of CHF rabbits. These data suggest antioxidant agents may be beneficial in CHF by down regulating AT1R expression and up regulating AT2R. The down stream pathway for AT1R regulation may be through JNK/MAPK and AP1.
INCREASED EXCITABILITY OF RVLM‐PROJECTING HYPOTHALAMIC PVN NEURONS IN ANGIOTENSIN II‐SALT HYPERTENSIVE RATS.
Authors: Chen Qing‐Hui , Dong Ying , Cardoso Leonardo , Shi Peng , Calderon Alfredo S , Andrade Mary A , Toney Glenn M
Published: June 2021

The contribution of the hypothalamic PVN to maintenance of SNA and high blood pressure (BP) in rats with Ang II‐salt HT was investigated in anesthetized rats by microinjecting the GABA‐A receptor agonist muscimol bilaterally into the PVN. Muscimol significantly (P<0.05) reduced renal SNA (51±5%) and MAP (27±10 mmHg) in rats with Ang II‐salt HT (n=3). In contrast, muscimol had no effect on renal SNA and produced a blunted fall of MAP (−15±5 mmHg, P<0.05) in normotensive rats (n=6). These data indicate an enhanced role for PVN neuronal activity in maintaining renal SNA and BP in rats with Ang II‐salt HT. Consistent with this interpretation, whole‐cell patch‐clamp recordings from brain slices revealed that excitability of RVLM‐projecting pre‐sympathetic PVN neurons from rats with Ang II‐salt HT was enhanced compared to neurons from normotensive controls. Indeed, graded current injections (50, 100, 150, 200 pA; 800 ms) evoked graded increases in action potential discharge frequency among neurons of both groups. Maximum discharge was evoked by 200 pA injections and averaged 47 ± 5 Hz in neurons from rats with Ang II‐salt HT (n=9). This response was significantly higher (P<0.05) than that of neurons from normotensive controls, which averaged 25 ± 3 Hz (n=12). We conclude that increased excitability of PVN‐RVLM neurons may contribute to sympathetic activation and maintenance of Ang II‐salt HT. Support: HL076312 (NCDC)
Hypercapnic ventilatory response of adult quail after chronic neonatal hypercapnia
Authors: Foster Jennifer A. , Broge Thomas A. , Atchley Dylan S. , Bavis Ryan W.
Published: June 2021

Embryonic exposure to 2% CO2 attenuates the hypercapnic ventilatory response (HCVR) of adult female Japanese quail (Coturnix japonica) by ~40% (Bavis & Kilgore, Respir. Physiol. 126:183–199, 2001); this treatment does not alter the HCVR of males. The present study investigated whether chronic hypercapnia during the neonatal period elicits similar developmental plasticity. Quail were exposed to 5% CO2 from hatching through 14–17 days of age; a control group was raised in air. Once sexually mature (6–9 weeks of age), the HCVR was measured by barometric plethysmography for quail acutely exposed to 6% CO2 (n=13–17 per sex per treatment group). Neonatal hypercapnia had no lasting effect on baseline ventilation. When HCVR is calculated as absolute ventilation during 6% CO2 or as the absolute increase in ventilation from baseline, HCVR was reduced by ~20% in females exposed to neonatal hypercapnia whereas males were unchanged (treatment × sex, P<0.05). Despite a similar trend, HCVR was not significantly reduced when calculated as a percentage of baseline ventilation or when ventilation is normalized to metabolic rate. Together, these data suggest that chronic neonatal hypercapnia has only minor effects on the adult HCVR in quail. Thus, the critical period for this developmental plasticity appears to lie primarily within the embryonic period for this precocial species. Supported by NIH grant P20 RR‐016463 (Maine INBRE).