bluuebunny/crossref_metadata_2025 huggingface dataset:
A Longitudinal study of the expression of Follicle Stimulating Hormone in Prepubertal Mice
Authors: Ward Linlei , Richter‐Maze Jennifer , Maze TD
Published: June 2021

Follicle‐stimulating hormone (FSH) is a glycoprotein secreted from the anterior pituitary. Like other glycoproteins, FSH exists as a mixture of isoforms that vary in the number and type of sugar groups. Observed changes in FSH isoforms during critical reproductive events, such as puberty onset, suggest that different combinations of FSH isoforms influence reproduction differently. It has also been suggested that all FSH isoforms may not equally bind to antibody based assays. In order to establish a working model, a study was performed using pre‐pubertal mice to determine at what age FSH mRNA expression begins. Gene expression was determined for the alpha, FSH beta, LH beta, and TSH beta subunits in five‐, ten‐, fifteen‐, twenty‐, thirty‐, and sixty‐day‐old mice. All subunits were expressed in all age groups indicating that FSH expression begins very early in development. However, despite the expression of mRNA, morphological studies do not reflect an active form of FSH is initiating gonad development, hormone production, or gametogenesis. Early expression of FSH may be critical to establish a hormonal milieu that leads to maturation of the gonads. Another possibility is that different isoforms of FSH determine its physiological significance during different metabolic states.
Continuous testosterone administration for 5 months reduces markers of bone turnover in older men
Authors: Dillon Edgar Lichar , Urban Randall J. , Angel James A. , Casperson Shanon L. , Paddon‐Jones Douglas , Sheffield‐Moore Melinda
Published: June 2021

Men and women undergo a progressive reduction in bone mineral density (BMD) with advancing age. Maintenance of circulating testosterone concentrations is beneficial to BMD in older men. We investigated whether continuous and monthly cycled administration of testosterone improves markers of bone metabolism in older men with low normal endogenous testosterone.

Older men (68.92 ± 1.59 yrs, serum total testosterone 362 ± 23 ng/dL, n=13) completed a 5‐month randomized double blinded placebo controlled protocol. Subjects received either continuous testosterone (weekly injections, 100 mg testosterone‐enanthate) for 5 months (TE, n=4), monthly cycled testosterone/placebo treatment (MO, n=4), or weekly placebo (PL, n=5). Fasting blood and 24‐h urine was collected each month and DEXA scans were obtained at 0 and 5 months to assess changes in markers of bone turnover and BMD.

Serum n‐telopeptide decreased in TE and showed a trend for decrease in MO when compared to PL. Serum osteocalcin decreased significantly in TE but not in MO when compared to PL. There were no changes in urinary deoxypyrrodinoline, bone‐specific alkaline phosphatase, intact‐parathyroid hormone, or calcitonin in any of the groups. Pelvic BMD decreased in PL and MO but not in TE.

These data show beneficial effects of continuous testosterone therapy on markers of bone turnover in older men with low‐normal endogenous testosterone concentrations.
Inhibition of Adenosine Monophosphate‐activated Protein Kinase (AMPK) ameliorates the effects of Interferon gamma (IFN gamma) on epithelial barrier function in T84 cells
Authors: Scharl Michael , Barrett Kim E , McCole Declan F
Published: June 2021

IFNγ plays a major role in the pathogenesis of inflammatory bowel disease (IBD). Treatment of intestinal epithelial cells with IFNγ leads to decreased transepithelial resistance (TER), increased epithelial permeability and a downregulation of tight junction proteins. AMPK has been shown to reduce energy consuming processes during inflammation. Here, we investigated if AMPK is involved in IFNγ‐induced effects on epithelial barrier function.

Protein analysis was performed by Western blot. TER was assessed by voltohmeter and confirmed with Ussing chambers.

IFNγ increased AMPK phosphorylation in T84 cells (1000 U/ml, 6h). The AMPK inhibitor, Compound C (50 μM), reversed the IFNγ‐induced decrease in TER (p<0.001, n=5) across T84 monolayers at 72h treatment. In contrast, activation of AMPK with AICA‐Riboside‐5′‐Phosphate (1 mM, 24–72h) had no effect on TER. AMPK inhibition also prevented the IFNγ induced downregulation of the tight junction protein, occludin (p<0.05, n=3, 72h).

In summary, these data show involvement of AMPK in IFNγ‐induced alterations in epithelial integrity. However, AMPK activity alone is not sufficient to generate such effects. These findings indicate a novel role for AMPK in the regulation of epithelial barrier function, and may have implications for inflammatory conditions associated with metabolic stress, such as IBD.

Supported by the Crohn's and Colitis Foundation of America and NIH.
Alzheimer's Beta‐Secretase in Health and Disease
Authors: Farah Mohamed , Wong Philip C
Published: June 2021

The neuritic plaques of Alzheimer's Disease (AD) are comprised of extracellular deposits of amyloid‐β (Aβ) peptides surrounded by dystrophic neurites, reactive astrocytes, and microglia. The Aβ peptides are derived from the sequential processing of amyloid‐β precursor protein (APP) by the activities of two enzymes termed, β‐ and γ‐secretase. The identification of β‐site APP cleaving enzyme 1 (BACE1) as the long sought β‐secretase required for the cleavage of APP to generate Aβ stimulated intense drug discovery research programs to identify pharmacological inhibitors of β‐secretase for the treatment of AD. Recent identification of important signaling pathways impacted by BACE1 raise concerns regarding the potential mechanism‐based toxicities associated with pharmacological inhibition of this aspartyl protease. For example, our recent discovery that BACE1 is critical for cognitive, emotional and synaptic functions raised concerns regarding mechanism‐based side effects associated with pharmacological inhibition of BACE1. Moreover, recent studies indicate that BACE1, acting through the neuregulin 1 (NRG1) signaling pathway, is required for proper myelination of central and peripheral axons. However, it is not known whether these alterations are restricted to development. To determine the impact of deletion of BACE1 during adulthood and aging as well as for clarifying mechanisms of clearance and repair from Aβ‐induced toxic effects, we generated and characterized an inducible BACE1 transgenic mouse model in which BACE1 expression is temporally and spatially controlled (using a “tet‐off” regulatory system). Results derived from these studies will have important implications for development of rational therapy to attenuate Aβ amyloidosis in AD.
Hypoxia‐inducible factor‐1 alpha regulates the hepatic mitochodnrial ATPase inhibitory protein (IF1)
Authors: Huang Li‐Ju , Yang Rei‐Cheng
Published: June 2021

Sepsis and ensuring multiple organ failure continue to be the most common causes of death in intensive care units, despite various major advances in the treatment of sepsis. The liver is though to be a major organ responsible for the initiation of multiple organ failure during sepsis due to its central role in metabolism and host defense mechanism. Our previous results showed that the suppression of IF1 (mitochondrial ATPase inhibitory protein) expression and subsequent elevated mitochondrial F0F1‐ATPase activity might contribute to the bioenergetic failure in the liver during sepsis, and the influence of decreased transcriptional lever on IF1 might an important factor. However, little is known about the transcriptional regulation of IF1 gene. In this study, we speculated that IF1 gene expression is modulated by a hypoxia‐sensitive transcription factor (HIF‐1), which is one of the dominant transcriptional factors under hypoxic condition. The results showed that the increased level of HIF‐1α protein, a component of HIF‐1, was concomitant with IF1 protein augmentation under hypoxic condition or CoCl2 (HIF‐1α activator) treatment. On the contrary, decreased nuclear HIF‐1α protein and mRNA level were followed by reduced mitochondrial IF1 protein level and mRNA expression in late sepsis. Antisense oligonucleotide against HIF‐1α greatly decreased the IF1 protein level in clone 9 epithelia cell line. In conclusion, these results are the first time to suggest that the HIF‐1α might play a crucial role in regulating the IF1 protein expression in late septic liver.
Altered Endothelin‐1 Signaling in the Activation of Cytosolic Phospholipase A2 (cPLA2) and Thromboxane A2 (TXA2) Productions in Kupffer Cells (KC) of the Prefibrotic Rat Liver: A Possible Mechanism in the Early Development of Portal Hypertension in Cirrhosis
Authors: Miller Andrew M , Zhang Jian X.
Published: June 2021

We sought to investigate altered ET‐1 signaling in cPLA2 activation in liver fibrosis. KC isolated from rats of 1 week bile duct ligation (BDL) or sham operation were treated with ET‐1 following pretreatment with inhibitors of Gi or Gq/G11 and intermediates in the signaling pathways. The activation of cPLA2 measured by phosphorylation and TXA2 productions in KC were determined. Inhibitions of cPLA2 with AACOCF3 or [Ca2+]i chelation with BAPTA‐AM, respectively, completely abolished ET‐1 induced releases of TXA2 from KC of both Sham and BDL, indicating that KC productions of TXA2 in response to ET‐1 are mediated primarily by Ca2+ dependent cPLA2. Inhibitions of MEK1/2‐ERK1/2 pathway with PD98059 or Gq signaling with GP2A, respectively, completely in Sham but only partially in BDL abrogated releases of TXA2 in ET‐1 stimulated KC. The inhibition of p38 MAPK with SB203580 or the blockade of Gi using pertussis toxin had no effect in Sham while markedly abolished ET‐1 induced releases of TXA2 in BDL. In addition, BDL resulted in a significant increase in Gαi but decrease in Gqα and G11α compared to Sham. While BDL caused no significant change in the phosphorylation of ERK1/2 in KC in response to ET‐1, it greatly enhanced the phosphorylation of p38 MAPK compared to Sham. All data suggest that ET‐1 signaling in cPLA2 activation may shift from Gq/G11 mediated activations of MEK12‐ERK1/2 to Gi mediated activations of p38 MAPK in KC in response to fibrosis.
Elevated circulating and endogenous lipids increase CREBH gene expression in vivo
Authors: Gentile Christopher L , Wang Dong , Wei Rosie , Pagliassotti Michael J
Published: June 2021

CREBH is a liver‐specific transcription factor that is activated by endoplasmic reticulum (ER) stress and required for C‐reactive protein (CRP) expression. We examined whether lipids regulate CREBH and CRP gene expression in vivo. A pancreatic clamp was performed in rats in which insulin and glucose were replaced at basal levels in the absence (CON, n=5) or presence of Liposyn (LIP, n=5) infusion. Free FA were increased ~4‐fold (p<0.05) in LIP throughout the study. CREBH mRNA was increased ~3‐fold in LIP vs CON. ER stress and CRP mRNA were not increased in CON or LIP. We next examined CREBH expression in dietary models of hepatic steatosis. Rats were fed a control, high starch diet (STD, n=4) or one of two diets that produce hepatic steatosis, a high‐sucrose diet (HSD, n=4) or high fat diet (HFD, n=4) for 4 wks. CREBH mRNA was increased ~2.5 fold in HSD and HFD vs STD. ER stress and CRP mRNA were increased (~2.5 fold) in HSD only. In conclusion, CREBH gene expression is regulated by circulating and hepatic lipids and CRP is associated with hepatic ER stress. Supported by DK 072017
THE MITOCHONDRIAL PERMEABILITY TRANSITION (MPT) AFTER LIVER ISCHEMIA AND REPERFUSION: ROLE OF C‐JUN N‐TERMINAL KINASE 2 (JNK2)
Authors: Theruvath Tom P , Snoddy Mark , Zhong Zhi , Lemasters John J
Published: June 2021

Here, we tested the hypothesis that JNK2 plays a role in promoting MPT onset and liver damage after hepatic ischemia and reperfusion (I/R). Methods. Wildtype (WT) and JNK2 knockout (JNK2 KO) mice underwent 70% liver ischemia for 1 h followed by reperfusion. After 8 h, hepatocyte injury was assessed by serum ALT, necrosis, and apoptosis at 8 h after reperfusion. Further to measure mitochondrial function, mice were anesthetized after 4 h, re‐laparotomized and infused with rhodamine 123 and propidium iodide (PI) to monitor mitochondrial membrane potential and loss of cell viability, respectively, using intravital laser scanning confocal/multiphoton microscopy. Results. After sham operation, wildtype and JNK‐2 KO mice were not different in ALT, necrosis, and apoptosis. After I/R, ALT values increased to 23,188 ± 2494 U/L in WT mice vs. 14,354 ± 3244 U/L in JNK2 KO mice (p<0.05). Histology demonstrated a comparable decrease in necrosis from 57% in WT to 35% in JNK2 KO mice (p<0.05). TUNEL staining revealed no difference in apoptosis between WT and JNK2 KO mice after I/R. At 4 h after I/R, mitochondrial depolarization in viable hepatocytes of livers of WT mice was 82 ± 5% vs. 43 ± 3% in JNK2 KO mice (p<0.05). Cell killing was 6 ± 2% in WT vs. 2.7 ± 2% in JNK2 KO mice (ns). In conclusion, JNK2 contributes to liver injury and cell death after I/R. Injury is associated with increased mitochondrial depolarization, a consequence of the MPT.
Inhibition of HIV‐1 Transcription by DpT‐based Iron Chelators
Authors: Debebe Zufan Kebede , Niu Xiaomei , Robinson Dillon , Richardson Des , Jerebtsova Marina , Ray Patricio , Gordeuk Victor , Nekhai Sergei
Published: June 2021

HIV‐1 transcription is activated by Tat protein, which recruits transcriptional co‐activators to the HIV‐1 promoter. Our recent studies show that CDK2 participates in HIV‐1 transcription by phosphorylating Tat protein. Thus inhibition of CDK2 might present a new approach to inhibit HIV‐1 transcription. We recently showed that iron chelators, 311 and ICL670, inhibited CDK2 activity and HIV‐1 transcription in CEM‐T cells and 293T cells [1]. Recently, a group of novel di‐2‐pyridylketone thiosemicarbazone (DpT)‐based iron chelators were shown to exhibit marked antiproliferative efficacy in vitro and anti‐tumor activity in vivo [2]. In this study we analyzed several DpT‐based chelators, Bp4aT, Bp4eT and Dp44mT for the inhibition of HIV‐1 transcription and evaluated their cellular toxicity. Our results show that all three chelators inhibited HIV‐1 transcription with approximately micromolar IC50s in CEM‐GFP cells infected with Adeno‐Tat, and 293T cells transfected with HIV‐1 LTR‐LacZ and Tat‐expressing plasmids. Analysis of cellular toxicity showed that iron chelator, Bp4eT was 3–5 less toxic as compared to Bp4aT or Dp44mT. Thus Bp4eT is the most suitable candidate as a potential HIV‐1 inhibitor.
Lactational hormones increase expression of milk proteins but do not enhance uptake of copper by mammary epithelial cells
Authors: Kim Stephen , Kassissa Christine , Kidane Theodros Z. , Ackland Leigh , Mercer Julian F.B. , Linder Maria C.
Published: June 2021

In lactation, a large portion of ionic copper entering the blood is diverted from the liver and kidney to the mammary gland and rapidly enters the milk. To determine whether this increased uptake of copper by the mammary gland might be due to the increased expression of specific copper transporters, we examined the response of the mammary epithelial cell to lactational hormones, using the human breast epithelial model (PMC42 cells) as well as cultured mouse mammary explants. Although the hormones induced increased expression of the milk protein, casein (as determined by Western blotting), uptake of 64Cu delivered on alpha‐2‐macroglobulin, albumin, or the di‐histidine complex was not enhanced. Manipulation of the Matrigel concentration to grow the PMC42 cells as organoids rather than monolayers also made not difference to rates of copper uptake. Expression of CTR1 was not affected by hormone treatment. Our findings suggest that the increased number and volume of the epithelial cells occurring in lactation, rather than changes in uptake capacity per cell, account for the increased flow of copper into the mammary gland during the suckling period. Indeed, we found that uptake of tracer 64Cu by mouse mammary gland increased in proportion to its weight already in gestation. Supported by PHS Grant No. RO1 HD 46949.
A role for the divalent metal‐ion transporter (DMT1) is doubtful in the mechanism by which calcium‐channel blockers reverse iron overload
Authors: Mackenzie Bryan , Shawki Ali , Ghio Andrew J , Stonehuerner Jacqueline D , Zhao Lin , Ghadersohi Saied , Garrick Laura M , Garrick Michael D
Published: June 2021

Fueling speculation about a connection between transferrin‐independent Fe uptake and Ca channels, Ludwiczek et al (Nat Med 13, 448 [2007]) concluded that dihydropyridine‐type Ca‐channel blockers (e.g. nifedipine) can reverse Fe overload by stimulating DMT1 activity and mobilizing tissue Fe. Since the paper disagrees with two others (Biochem J 398, 539 [2006]; Biochem J 403, 59 [2007]) on the transport activity of DMT1 isoforms, studies were undertaken to resolve that issue and explore the effect of nifedipine on DMT1, using doxycycline‐induced expression of DMT1 in permanently‐transfected HEK293 cell lines and expression of DMT1 in Xenopus oocytes. We confirmed our cited studies that the isoforms have very similar activities. Nifedipine did not stimulate DMT1 activity in any assay. Morgan & Savigni (Biochem Pharmacol 51, 1701 [1996]) observed that photodegraded nifedipine (PDN) stimulated Fe uptake by erythrocytes and suggested that PDN was an Fe‐specific ionophore. We confirmed this property of PDN in our preparations and found that it was independent of DMT1 since: (1) PDN stimulated uptake of Fe, but not of Mn, whereas each are DMT1 substrates; (2) The effect of PDN on Fe uptake was less apparent when cells over‐expressed DMT1 than when DMT1 levels were lower or minimal. The report by Ludwiczek et al is of clinical significance because of the need for ways to increase Fe excretion, but how nifedipine leads to such an increase remains unclear.
Molecular cloning, distribution and developmental regulation of porcine GLUT2 mRNA in small intestine
Authors: Huang Zhiyi , Feng Dingyuan , Zou Shigeng , Zhi Aimin , Zuo Jianjun
Published: June 2021

Molecular cloning, tissue distribution and developmental regulation of porcine GLUT2 (glucose transporter 2) mRNA were conducted in small intestine. GLUT2 cDNA is 2051 bp long, the predicted amino acid sequence revealed that it is highly conserved among vertebrate species. Animal tissue samples were collected from 35 pigs at different developmental stages: sucking (day 1, 7 and 26), postweaning (day 30) and growing (day 60, 90 and 150). The results showed that GLUT2 mRNA was detected at highest level in porcine liver, at moderate levels in the small intestine, kidney, and at low levels in the brain, lung, muscle and heart. In the small intestine, the highest level is in the jejunum. After weaning, the three small intestinal sements took on a similar pattern, GLUT2 mRNA increased through post‐weaning (d30, 2 days post‐weaning) and peaked in growing period (d90), and then decreased in finishing period (d150). In conclusion, the mRNA expression of GLUT2 was not only differentially regulated by age but also differentially distributed along the small intestine of piglets at different stages, which may be related to intestinal luminal substrate concentrations. This study was supported by the National Basic Research Program of China (Project No: 2004CB117501) and National Natural Science Foundation of China (Project No: 30671529).
A new population of bone marrow cells restores lung growth in a model of Bronchopulmonary Dysplasia (BPD)
Authors: Balasubramaniam Vivek , Ryan Sharon L , Seedorf Gregory J , Markham Neil E , Yoder Mervin C , Abman Steven H
Published: June 2021

BPD is characterized by impaired lung growth that persists in adulthood. We have shown neonatal hyperoxia reduces bone marrow progenitor cells & causes abnormal lung structure in mice. The role of progenitor cells in lung growth is controversial. We isolated a new population of bone marrow derived angiogenic cells (BMDAC). We hypothesize that BMDACs can home & engraft in the lung, & promote vascular & alveolar growth after neonatal hyperoxic lung injury.

Methods: BMDACs from transgenic Tie2‐GFP mice, were cultured on a feeder cell layer, & characterized by FACS. Neonatal mice were exposed to hyperoxia (FiO2=0.8) X 10 days. BMDAC (10^5 cells) were injected into the right ventricle prior to room air recovery X 10 days. Lung morphometry & immunofluorescence was done.

Results: BMDAC displayed a phenotype of a myeloid progenitor: CD45+/CD34‐/CD38+/CD11b‐/CXCR4+/Ly6c‐/F4/80+. Hyperoxia impaired lung structure, which persisted in room air recovery in controls. BMDAC treatment improved septation by 78% & vessel density by 134%(p<0.01), equal to room air raised mice. BMDAC engraftment was 12%, & localized to vessel wall & interstitium, in association with Type II cells. BMADCs did not co‐express smooth muscle, endothelial, epithelial or pericyte markers. We conclude BMDACs are a novel cell population that home to sites of injury in the infant lung & stimulate alveolar & vascular growth after neonatal hyperoxia.
Cysteine mutagenesis to study the structure of claudin‐2 paracellular pores
Authors: Angelow Susanne , Yu Alan S.L.
Published: June 2021

Claudins are tight junction proteins that control passive paracellular transport of small ions across epithelia. Claudin‐2 is expressed in proximal renal tubules which partly re‐absorb Na(+) via the paracellular pathway and it forms cation‐selective paracellular pores in high resistance cell lines. To test the hypothesis that amino acid residues in the 1st extracellular loop line the paracellular pore, we used cysteine mutagenesis of Y35, H57, D65 and I66 in claudin‐2 and expressed these mutants in stable transfected MDCK I cells. H57C and D65C showed a partial loss of cation permeability (50% of P(Na+) of wild‐type claudin‐2), suggesting that these residues may participate in cation permeation. D65C also exhibited marked loss of charge and size selectivity and appeared as a dimer on non‐reducing polyacrylamide gels, indicating that conformational changes resulting from intermolecular disulfide bonding disrupted the pore structure. Permeability and selectivity of Y35C and I66C were preserved. Addition of cationic methanethiosulfonate reagents to I66C caused a prompt decrease in conductance of up to 35%, suggesting that this residue may line the pore lumen and be accessible to extracellular cations. By applying methods used to investigate transcellular ion channels, we are beginning to map the position of key residues within the paracellular pore and develop MTS reagents as potential pore blocking tools.
Effect of diazoxide on the mitochondrial respiration of skeletal muscle
Authors: Montoya‐Pérez Rocío , Trujillo Xóchitl , Huerta Miguel , Saavedra‐Molina Alfredo
Published: June 2021

Mitochondrial KATP channels were described by Inoue (1991) by patch clamp techniques on isolated mitochondria of liver. Later on were described on mitochondria of heart and brain. Potassium channels are inhibited by sulfonylurea and activated by potassium channel openers (KCOs). It has been reported that diazoxide is a potent selective opener of the mitochondrial KATP channel (Garlid et al., 1997; Sato et al., 1998) in a range of 10 to 50 μM (Kowaltowski et al., 2001). Also, it has been proposed that this drug can act on other proteins of the respiratory chain, like succinate dehydrogenase enzyme, with concentrations up the 100 μM (Hanley et al., 2002). In the present work we investigated the effect of diazoxide (100 – 500 μM) on mitochondrial respiration of skeletal muscle of chick, using different substrates. We observed a dose‐dependent inhibition using glutamate‐malate and succinate as substrates for complex I and II, respectively (40% of inhibition). The dose used is higher of those reported for cardiac and skeletal muscle, reason why we conclude that mito‐KATP of skeletal muscle is less sensitive to KCOs and we do not discard that the effect could be do to the effect on the membrane potential or calcium uptake. It is required a more complete analysis to contribute a concrete conclusion. Supported by the grants from FRABA to X.T., COECYT, Fondos Mixtos Gob. Michoacán‐CONACYT (64277) and CIC‐UMSNH (2.16) to S‐M.A.
WNK3 enhances TRPV5‐mediated calcium transport via a kinase‐dependent pathway
Authors: Zhang Wei , Na Tao , Peng Ji‐Bin
Published: June 2021

WNK3, a member of the With No Lysine (K) family of protein serine/threonine kinases, was shown to regulate members of the SLC12 family of cation‐chloride cotransporters and the renal outer medullary K+ channel ROMK. To evaluate the effect of WNK3 on TRPV5, a renal epithelial Ca2+ channel that serves as a gatekeeper for active Ca2+ reabsorption, WNK3 with TRPV5 were co‐expressed in X. laevis oocytes and the function and expression of TRPV5 were subsequently examined. An 80.6±11.4% increase in TRPV5‐mediated Ca2+ uptake was observed when WNK3 was co‐expressed. Similar increase in TRPV5‐mediated Na+ current was observed with voltage clamp technique. WNK3 also enhanced Ca2+ influx mediated by TRPV6, which is the closest homologue of TRPV5 that mediated active intestinal Ca2+ absorption. The positive regulatory effect was abolished by the kinase‐inactivating D294A mutation in WNK3, indicating a kinase‐dependent mechanism. The fully glycosylated TRPV5 that appears at the plasma membrane was increased by WNK3, indicating that WNK3 increases TRPV5 expression at the cell surface. In the presence of brefeldin A, WNK3 slowed the declining of TRPV5‐mediated Ca2+ uptake over a period of 6 hours, suggesting that WNK3 stabilizes TRPV5 at the plasma membrane. In contrast, WNK3 exerted no significant effect on the delivery of TRPV5 to the plasma membrane from its intracellular pool. These results indicate that WNK3 is a positive regulator of transcellular Ca2+ transport pathway. Supported by NIH grant DK072154.
Stimulation of β3–adrenoceptors activates spontaneous transient outward BK currents and causes relaxation in rat urinary bladder smooth muscle
Authors: Cui Xiangli , Hristov Kiril L. , Kellett Whitney F. , Brown Sean M. , Petkov Georgi V.
Published: June 2021

The role of large conductance Ca2+‐activated K+ (BK) channels in the β3‐adrenoceptor (β3‐AR)‐induced relaxation in rat urinary bladder smooth muscle (UBSM) was studied. BRL 37344, a specific β3‐AR agonist, inhibits spontaneous contractions of isolated UBSM strips. Iberiotoxin, a specific BK channel inhibitor, shifts the BRL 37344 concentration‐response curves for contraction amplitude, muscle force, and tone to the right. Freshly isolated UBSM cells and the perforated patch‐clamp technique were used to determine further the effect of β3‐AR stimulation on the BK channel activity. BRL 37344 increased spontaneous transient outward BK current (STOC) frequency by 46%. In the presence of ryanodine and nifedipine, the single BK channel amplitude and open probability recorded in cell‐attached mode at Vh=0 mV as well as the current‐voltage relationship in response to depolarization steps were not affected by BRL37344. In current‐clamp mode, BRL 37344 caused membrane potential hyperpolarization of about 3 mV. The BRL 37344‐induced hyperpolarization was eliminated by application of iberiotoxin or 1 mM tetraethylammonium. This study provides evidence that in rat UBSM, β3‐ARs and BK channels are functionally coupled so that stimulation of β3‐AR leads to an increase in the BK channel STOC frequency, which causes membrane hyperpolarization and relaxation. Supported by DK070909 to G.V.P.
Effects of nitric oxide levels on hIK1 expression
Authors: Mague Alissa Renee , Jones Heather Marie
Published: June 2021

The calcium (Ca2+)‐activated potassium (K+) channel, hIK1, is an epithelial ion channel having roles in many essential physiological functions including: ion transport, cell volume regulation and endothelial cell hyperpolarization. Nitric oxide (NO) is a potent vasodilator which allows for relaxation of the smooth muscle surrounding the capillary endothelial cells. Since both hIK1 and NO are involved in endothelial cell vaso‐regulation, we determined the effect NO has on hIK1 protein expression. The effects of the NO donors: SNAP and PAPNONOate and the NOS inhibitor, L‐NMNA were examined. Human embryonic kidney (HEK) cells transfected with hIK1 protein were treated with NO agents 18 hours prior to experimentation. Cells were lysed, total protein collected, and run on SDS‐PAGE gel. Antibodies were used to detect total hIK1 protein in treated verses untreated HEK cells. Results showed that elevated NO levels increased total hIK1 protein expression when compared to untreated cells. Lowering NO levels decreased total protein expression. Localization of hIK1 at the cell surface is regulated and may be altered by various signaling molecules. The inhibitor of NOS decreased hIK1 levels at the plasma membrane as assessed by cell surface immunoprecipitation. These results suggest that NO plays an important role in the regulation of hIK1 expression which may be related to it's role in endothelial cell function.
Substitutions of a pair of histidine residues within the extracellular loop of alpha subunit of the epithelial sodium channel suppress sodium self‐inhibition
Authors: Sheng Shaohu , Maarouf Ahmad B. , Kleyman Thomas R.
Published: June 2021

Epithelial Na+ channels (ENaC) mediate apical Na+ transport and play an essential role in body fluid volume homeostasis. Recent studies have demonstrated a key role of the extracellular Na+ concentration in controlling the open probability of ENaC through Na+ self‐inhibition. Here we investigated the role of a pair of adjacent His residues within the extracellular loop in Na+ self‐inhibition. The His325 and His326 of mouse αENaC were mutated to di‐Ala residues. The Na+ self‐inhibition responses for wild type and mutant channels expressed in Xenopus oocytes were examined by two electrode voltage clamp. A significantly suppressed (50%) and slowed (65%) Na+ self‐inhibition was observed in oocytes expressing αH325A‐H326Aβγ, compared to the wild type. Based on the recently published structure of the chicken acid sensing ion channel 1 (cASIC1) and a sequence alignment with αENaC, the di‐His residues are predicted to be located at the C‐terminus of β4 within the β‐ball domain. Interestingly, residue Arg191 of cASIC1, which is adjacent to the sequence homologous to HH of αENaC, directly interacts with a residue (Asp350) within the thumb domain that is predicted to bind proton. Based on our results and the cASIC1 structure, we propose that the di‐His residues interact with the thumb domain and the intrasubunit interaction is involved in the conformational changes induced by the binding of extracellular Na+. (Supported by NIH DK54354)
Molecular and Functional Characterization of Murine Bestrophin 1 Cloned from Heart
Authors: O’Driscoll Kate Eileen , Leblanc Normand , Britton Fiona C
Published: June 2021

Calcium‐activated chloride channels (ClCa) are widely distributed in cardiac tissues and play important roles in the regulation of cardiac excitability. However, the molecular identity of this channel remains unknown. Bestrophins were recently shown to form ClCa when expressed heterologously. We hypothesize that Bestrophins are candidates for the calcium‐activated chloride current (IClCa) in cardiac tissues. We demonstrate that several members of this family are expressed in heart. Previous studies have reported the functional expression of two members of the murine Bestrophin family, mBest2 and mBest3. Here we report the functional characterization of mBest1. We isolated the cDNA transcript of mBest1 from mouse heart and cloned it into a mammalian expression vector so that functional analysis could be performed to determine if it encodes a ClCa. Whole‐cell patch clamp experiments with HEK cells transfected with mBest1 revealed a calcium sensitive, time independent chloride current. This DIDS‐ and niflumic acid‐sensitive current displayed slight voltage dependence and exhibited a lyotropic sequence of anion permeability, SCN−>I−>Cl−. Our data contributes to the characterization of the biophysical properties of Bestrophins and helps further the investigation into the molecular candidate of cardiac IClCa.
Effect of dietary flavonoids on hormone‐stimulated ion transport in renal principal cells
Authors: Creason Lacey Beth , Muchatuta Monalisa Ndanatsei , Platt Laura Keese , Blazer‐Yost Bonnie L
Published: June 2021

Flavonoids are a class of compounds found in plants such as parsley, cocoa, soybeans, tea, and grapes. Flavonoids have anti‐oxidant properties, can act as retroviral inhibitors, and have cardiovascular benefits. We hypothesize that the cardiovascular benefits are, in part, due to an effect on salt and water homeostasis via the renal epithelial Na+ channel (ENaC). Quercetin, apigenin, and genistein are the dietary flavonoids of interest. Insulin and aldosterone regulate ENaC through a PI3‐kinase dependant pathway. A PI3‐kinase inhibitor, LY294002, was designed from the quercetin structure. Therefore, we hypothesize that flavonoids may inhibit ENaC activity. We used electrophysiological techniques to study the effects of flavonoids on ion transport in the mouse principle cells of the kidney cortical collecting duct (mpkCCD) cell line. All three flavonoids lower basal ion transport in mpkCDD cells. Neither genistein nor apigenin inhibit insulin stimulated ion transport. Following ADH stimulation, both flavonoids inhibit anion secretion via CFTR, but have no statistical effect on ADH‐stimulated Na+ reabsorption. In summary, in mpkCCD cells, the flavonoids lower basal, amiloride sensitive Na+ transport, but do not alter peptide‐hormone stimulated Na+ reabsorption. The flavonoids do, however, inhibit ADH‐stimulated anion secretion.

Funding: IUPUI Undergraduate Research Opportunities Program.
TGF‐β1 Effect on Cortical Collecting Duct Cells
Authors: Feng Zhuang , Somanna Naveen K , Hamm Lee L , Hering‐Smith Kathleen S
Published: June 2021

ENaC in collecting duct (CD) cells is involved in the regulation of Na balance and blood pressure. Prostasin, an endogenous protease, has been shown to activate ENaC, which can be mimicked by trypsin. Basolateral (BL) TGF‐β1 has been shown to inhibit Na current (Ieq), mRNA expression of prostasin and ENaC subunits. The purpose of this study was to characterize the CD response to TGF‐β1, in particular the involvement of proteolytic processes and the paracellular pathway. M‐1 cells were studied on permeable supports. The activity of secreted serine proteases was measured by an amidolytic assay. BL TGF‐β1 inhibited Ieq in the range of 2–40 ng/ml. The most marked effect was observed at concentrations ≥20 ng/ml, with >60% inhibition at 24 hours. However, transepithelial resistance Rte also decreased significantly after 2 hours, and to > 50% inhibition at 24 hours, an effect not expected with inhibition of ENaC only. The effects of TGF‐β1 on Ieq and Rte were not reversed by trypsin. Proteolytic activity in the apical media was not altered by TGF‐β1. In conclusion, BL TGF‐β1 significantly decreases Na transport in CD cells but also increases paracellular permeability. The lack of response to trypsin in TGF‐β1 treated cells implies that the down‐regulation of prostasin is not the major factor in the inhibition of Na current by TGF‐β1.

Supported by research grants from VA and NIH.
Opposite effects of WNK3 and WNK4 upon the Slc26a9 anion transporter/channel.
Authors: Plata Consuelo , Vázquez Norma , F Romero Michael , Gamba Gerardo
Published: June 2021

Slc26a9 is an electrogenic nCl−/HCO3− exchanger with a large anion conductance that is mainly expressed in respiratory and gastrointestinal epithelial cells. It was recently shown that Slc26a9 is negatively regulated by the serine/threonine kinases WNK1, WNK3, and WNK4, and that these effects were not dependent upon the kinase activity. However, WNK3 and WNK4 have been shown to exhibit opposite effects in other chloride cotransporters. To gain insight to putative kinase regulation of Slc26a9, we evaluated the effect of protein kinases WNK3 and WNK4 on Slc26a9‐mediated anion transport using 36Cl− uptake in Xenopus laevis oocytes. Oocytes were injected with Slc26a9‐cRNA alone or together with WNK4 or WNK3‐cRNA. We observed that WNK4 caused a ~30% decrease in 36Cl− uptake by Slc26a9. This inhibition was not observed with co‐injection of D318A‐WNK4 which renders WNK4 catalytically inactive. Co‐injection of oocytes with Slc26a9 and WNK3 increased Slc26a9 activity by ~40% above baseline. This increased Slc26a9 activity by WNK3 was also kinase dependent, i.e., Slc26a9 activation was eliminated by D294A‐WNK3 which renders WNK3 catalytically inactive. Thus, our data indicate that WNK3 is a positive regulator of transport Cl− and WNK4 is a negative regulator of Cl− transport by the Slc26a9 anion transporter/channel. Furthermore, this Slc26a9 transport regulation is dependent on kinase activity of WNK3 and WNK4.
Interaction of the multivalent PDZ domain protein PDZK1 with novel liver‐specific organic anion transporter 7 (OAT7)
Authors: Anzai Naohiko , Shin Ho Jung , Miyao Kazuhiro , Endou Hitoshi
Published: June 2021

Hepatic organic anion transporters play an important role in the elimination of toxic substances and in the turnover of endogenous biomolecules. Recently, we functionally characterized a novel organic anion transporter OAT7 (SLC22A9) exclusively expressed in human liver. OAT7 mediated high affinity transport of sulfate‐conjugated steroids, estrone sulfate (ES) and DHEA sulfate using short chain fatty acid butyrate as a counterion (Shin et al., Hepatology, 2007). The C‐terminal domain of OAT7 is exposed to the cytoplasmic compartment and contains PDZ motif, one of the famous protein‐protein interaction modules, suggesting that it may interact with PDZ proteins. In the present study, we performed yeast two‐hybrid experiments against PDZ protein containing prey vectors using the C‐terminus of OAT7 as bait. As a result, we found that OAT7 C‐terminus interacted with the multivalent PDZ domain‐containing protein, PDZK1. Deletion of the OAT7 C‐terminal PDZ motif abolished the interaction with PDZK1 in the yeast two‐hybrid system. In addition, the third PDZ domains of PDZK1 associate strongly with the OAT7 C‐terminus. Cotransfection of OAT7 and PDZK1 into the HEK293 cells enhanced [3H]ES uptake via OAT7 (1.2‐folds). The elucidation of these interactions may lead to the further understanding of the function and regulation of organic anion transports in human liver.
Effect of gadopentetate dimeglumine administration on intestinal permeability to glucose after traumatic brain injury
Authors: Santos Alejandro , Gonçalves Pedro , Araújo João R. , Martel Fátima
Published: June 2021

We studied the effects of traumatic brain injury (TBI) and gadopentetate dimeglumine (GAD) administration on intestinal glucose absorption. Male Wistar rats were i.p. anaesthetised with ketamine and medetomidine. Thirty minutes after TBI (closed head trauma model) one group of rats (TBIGAD, n = 7) was administered GAD (70 mg.kg−1i.v.) and another group (TBI, n = 7) received saline. Control groups (CONT, n=7) were sham operated and received no weight drop and GAD (n = 7) group was administered GAD. Flat sheets of jejunal tissue in Ussing chambers were used to study mucosal‐to‐serosal permeability (AP‐to‐BL Papp) to two glucose model substrates, 3H‐2‐deoxy‐D‐glucose (3H‐DG) and 3H‐3‐O‐methyl‐D‐glucose (3H‐OMG), and to 14C‐sorbitol. Student's t‐test was used. TBI induced an increase in the AP‐to‐BL Papp to 3H‐DG. After a 100‐min perfusion of the jejunum, the AP‐to‐BL Papp to 3H‐DG in TBI rats was almost 70% higher than in control rats. There was no change in the AP‐to‐BL Papp to neither 3H‐OMG nor 14C‐sorbitol. Interestingly enough, GAD was able to prevent the increase in the AP‐to‐BL Papp to 3H‐DG observed after TBI. Our results suggest that GLUT2 is activated by TBI (as the Papp to DG, a GLUT2 substrate, was increased) and SGLT1 is inhibited by TBI (as the Papp to OMG, a GLUT2 and SGLT1 substrate, remained unchanged). GAD prevented these changes associated with TBI. Acknowledgements: FCT ‐ PTDC/SAU‐FCF/67805/2006
Cold Stress Triggered Alterations in Gene Expression in Rat Adrenal Medulla
Authors: Sabban Esther L , Liu Xiaoping , Kvetnansky Richard , Serova Lidia , Sollas Anne , Zilinksi Roni
Published: June 2021

Prolonged cold stress elicits sensitization of various neuronal systems, including the adrenal medulla (AM) to a novel or heterotypic stressor. To understand the molecular mechanism, a rat cDNA microarray (Affymetrix RAE 230 2.0) was used to analyze the gene expression profile of AM of rats exposed to cold stress (4°C) for 1, 7 or 28 days compared to unstressed controls. The number of transcripts significantly changed >1.5 fold were similar during the cold stress with 46, 69 and 58 up‐regulated and 28, 32, and 25 down‐regulated on days 1, 7 and 28 respectively. Less than half of the up‐regulated, and 20% of the down‐regulated transcripts were changed throughout the 28 days of cold. The greatest increase was seen in those transcripts responsible for proteins involved in gene transcription, secretion/neuropetides, signaling pathways and metabolism and the greatest decrease encompassed these categories as well as those related to channels and chemokines. We confirmed the induction of CREM, bradykinin receptor 2, phospholipase C epsilon, and two factors involved in synaptic vesicle function, VMAT2 and EH domain containing protein. Based on the results, we speculated that up regulation of synaptic vesicle function and/or changes in specific signaling pathways may mediate the sensitization to stress in the AM with cold exposure.
Sterol Regulatory Transcription Factor‐1: Key regulator of Fasting Response in the Adipose Tissue in Pigs?
Authors: Lkhagvadorj Sender , Qu Long , Cai Weiguo , Couture Oliver P , Wang Yanfang , Barb Richard C , Hausman Gary J , Rekaya Romdhane , Nettleton Daniel S , Anderson Lloyd L , Dekkers Jack C , Tuggle Christopher K
Published: June 2021

The genetic mechanisms controlling appetite and feeding behaviors are not well understood. In this study, transcriptional profiling was used to identify porcine genes and pathways that respond to a fasting treatment or to a missense mutation (D298N) in the melanocortin‐4 receptor (MC4R) gene, which has been associated with increased growth and feed efficiency. Prepubertal gilts (n=24) homozygous for D298N MC4R or wildtype were either fed ad lib. or fasted for 3 days in a completely randomized block design with 2×2 factorial treatment structure. The Affymetrix Porcine Genome was used to profile gene expression of liver and subcutaneous fat. Due to fasting, 7,029 genes in adipose tissue and 1,831 genes in liver were declared differentially expressed (q<0.05), but an effect of MC4R was not observed on expression in both tissues under same criterion. Pathway analyses indicated that 23 of the tested set of 298 genes that were down‐regulated (p<0.05, fold change>2) in the adipose tissue due to fasting were directly regulated by sterol regulatory element binding transcription factor‐1 (SREBF1). We confirmed the expression levels of SREBF1 and of its targets such as fatty acid synthase, aconitase‐1, acetyl CoA carboxylase alpha, and acetyl CoA synthase in the adipose tissue by qPCR. Results indicate that SREBF1 may play a key role in determining the pathways that respond to fasting in pigs. (USDA‐NRI‐2005‐3560415618).
Tx2‐6 toxin from Phoneutria nigriventer spider improves relaxation induced by electrical stimulation of rat cavernosum strips
Authors: Nunes Kenia Pedrosa , Cordeiro Marta Nascimento , Richardson Michael , Tostes Rita , De Lima Maria Elena , Webb R. Clinton , Leite Romulo
Published: June 2021

Background: Accidents involving the spider P. nigriventer are characterized by different symptoms including priapism. Some toxins from poisonous animals exert their toxic effects by modifying the properties of ion channels. We have previously demonstrated that Tx2‐6, a peptide extracted from the venom of P. nigriventer, improves erectile function in anesthetized rats. Here we investigated the mechanism involved in the Tx2‐6 potentiation of penile erection.

Methods: Rat cavernosum strips were incubated with bretylium (3 x 10–5M) and contracted with phenylephrine (10–5 M). Relaxation responses were evoked by electrical field stimulation (EFS) or sodium nitroprusside (SNP) before and after 5 min incubation with Tx2–6 (10–8 M). The effect of Tx2‐6 on relaxation induced by EFS was also tested in the presence of atropine (10–6 M), conotoxin GVIA (10–6 M) or nifedipine (10–7 M).

Results: Relaxation induced by SNP was not affected by Tx2‐6. EFS‐induced relaxation was significantly potentiated by Tx2‐6 and this effect was not affected by atropine, whereas blockade of N‐ or L‐Type Ca+2 channels abolished the potentiation of the relaxation induced by Tx2‐6.

Conclusion: Tx2‐6 potentiates the relaxation of cavernosum tissue via Na+ and Ca+2 channels, facilitating the nitric oxide/cGMP pathway. Tx2‐6 has great potential for development of drugs to treat erectile dysfunction.

SUPPORT: CNPq, FAPEMIG, NIH
The use of non‐linear analyses in the characterization of hypertension and associated autonomic dysfunction
Authors: Bartels Matthew N , Meersman Ronald E De , Donnelly Diane L , Ellythy Marzuk , Rockland Ronald H
Published: June 2021

To assess the relative merits of linear vs non‐linear analysis methods for detecting early autonomic changes in prehypertensive (PHT) subjects, we compared 24hr ambulatory blood pressure (BP) recordings for 6 PHT and 6 matched normotensive (NT) subjects. Along with linear techniques (BP variability, heart rate variability and baroreflex using the alpha method), we used non‐linear techniques (approximate entropy, detrended fluctuation analysis and return map/Poincare plot). Analysis was blinded. RESULTS: While standard linear methods did not discriminate between PHT and NT, non‐linear techniques showed significant differences. Approximate entropy analysis of systolic BP data exhibited higher values throughout the 24hr period (p < 0.05). Detrended fluctuation analysis of the daytime BP revealed elevated alpha‐1 of systolic BP in the PHT subjects (p < 0.05). The return map/Poincare plot of the 2 groups failed to reveal any clearly statistically significant changes. In summary, non‐linear analysis tools appeared superior to linear analyses in detecting PHT from NT subjects, and should be considered when dealing with non stationary and difficult data sets. Support: VIDDA foundation.
Essential role for inositol 1,4,5‐trisphosphate receptor 1 (IP3R1) in UTP‐induced Ca2+ signal and diameter regulation in rat cerebral arteries
Authors: Zhao Guiling , Blaskova Eva , Adebiyi Ade , Jaggar Jonathan H.
Published: June 2021

Inositol 1,4,5‐trisphosphate receptors (IP3R) regulate Ca2+ release from intracellular stores and modulate physiological functions including cell proliferation and vascular contraction. Three subtypes of IP3Rs have been described, but expression profiles and physiological functions of specific isoforms in vascular myocytes are unclear. Using RT‐PCR, immunoblotting, and immunofluorescence, we found that all three IP3R isoforms (1–3) are expressed in cerebral artery myocytes. Real‐time PCR indicated that IP3R1 was the most abundant (~82% of total), with IP3R2 and 3 less highly expressed (6 and 12%, respectively). UTP‐induced Ca2+ waves in myocytes of arterial segments were reduced from 0.7±0.1 Hz to 0±0 and 0.07± 0.04 Hz by the IP3R blockers 2‐APB (100 μM) and xestospongin C (20 μM), respectively. A shRNA suppression vector decreased IP3R1 protein by ~53% and reduced UTP‐induced Ca2+ wave frequency from 0.44±0.13 to 0.13±0.05 Hz, when compared with a scrambled control vector. In contrast, IP3R1 knockdown did not alter Ca2+ wave frequency in the absence of UTP. IP3R1 suppression also decreased UTP‐induced vasoconstriction in pressurized (20 mmHg) arteries from 30±3 μm to 15±3 μm. These results indicate that IP3R1 is the predominant IP3R isoform expressed in rat cerebral artery myocytes and is essential for UTP‐induced Ca2+ signal modification and contraction.