Regulation of Na(+) reabsorption by the aldosterone-induced small G protein K-Ras2A

Xenopus laevis A6 cells were used as model epithelia to test the hypothesis that K-Ras2A is an aldosterone-induced protein necessary for steroid-regulated Na(+) transport. The possibility that increased K-Ras2A alone is sufficient to mimic aldosterone action on Na(+) transport also was tested. Aldosterone treatment increased K-Ras2A protein expression 2.8-fold within 4 h. Active Ras is membrane associated. After aldosterone treatment, 75% of K-Ras was localized to the plasma membrane compared with 25% in the absence of steroid. Aldosterone also increased the amount of active (phosphorylated) mitogen-activated protein kinase kinase likely through K-Ras2A signaling. Steroid-induced K-Ras2A protein levels and Na(+) transport were decreased with antisense K-ras2A oligonucleotides, showing that K-Ras2A is necessary for the natriferic actions of aldosterone. Aldosterone-induced Na(+) channel activity, was decreased from 0.40 to 0.09 by pretreatment with antisense ras oligonucleotide, implicating the luminal Na(+) channel as one final effector of Ras signaling. Overexpression of K-Ras2A increased Na(+) transport approximately 2.2-fold in the absence of aldosterone. These results suggest that aldosterone signals to the luminal Na(+) channel via multiple pathways and that K-Ras2A levels are limiting for a portion of the aldosterone-sensitive Na(+) transport.     

Quantum-dye labeled proteins for glycobiology: a viable nonradioactive alternative tracer

Quantum dye (QD), a macrocyclic europium-chelate, developed as a cytological marker, has never been used for quantitative applications. It would be ideal, however, if the same tracer can be used for both qualitative and quantitative purposes. We have labeled some lectins and neoglycoproteins with QD for the purpose of quantitative analyses in glycobiology, and tested its suitability in three different areas in glycobiology: (1) glycosyltransferase, (2) an animal lectin - mannose- binding protein, and (3) the Gal/GalNAc receptor of rat liver membrane. Usefulness of QD-labeled lectins was amply demonstrated by the quantification of galactosyltransferase activity using QD-soybean agglutinin and QD-RCA120 ( Ricinus communis agglutinin). We also showed that QD-labeled neoglycoproteins, QD-Man-BSA and QD-Gal-BSA, can replace radioiodinated counterparts in the binding assays of animal lectins (serum mannose binding protein and hepatic Gal/GalNAc receptor.) The advantage of QD and other europium labels is that it does not decay as radioiodides do. The long shelf-life results in more consistent results from repeated experiments.      

Apical ammonium inhibition of cAMP-stimulated secretion in T84 cells is bicarbonate dependent

Normal human colonic luminal (NH(4)(+)) concentration ([NH(4)(+)]) ranges from approximately 10 to 100 mM. However, the nature of the effects of NH(4)(+) on transport, as well as NH(4)(+) transport itself, in colonic epithelium is poorly understood. We elucidate here the effects of apical NH(4)(+) on cAMP-stimulated Cl(-) secretion in colonic T84 cells. In HEPES-buffered solutions, 10 mM apical NH(4)(+) had no significant effect on cAMP-stimulated current. In contrast, 10 mM apical NH(4)(+) reduced current within 5 min to 61 +/- 4% in the presence of 25 mM HCO(3)(-). Current inhibition was not simply due to an increase in extracellular K(+)-like cations, in that the current magnitude was 95 +/- 5% with 10 mM apical K(+) and 46 +/- 3% with 10 mM apical NH(4)(+) relative to that with 5 mM apical K(+). We previously demonstrated that inhibition of Cl(-) secretion by basolateral NH(4)(+) occurs in HCO(3)(-)-free conditions and exhibits anomalous mole fraction behavior. In contrast, apical NH(4)(+) inhibition of current in HCO(3)(-) buffer did not show anomalous mole fraction behavior and followed the absolute [NH(4)(+)] in K(+)-NH(4)(+) mixtures, where K(+) concentration + [NH(4)(+)] = 10 mM. The apical NH(4)(+) inhibitory effect was not prevented by 100 microM methazolamide, suggesting no role for apical carbonic anhydrase. However, apical NH(4)(+) inhibition of current was prevented by 10 min of pretreatment of the apical surface with 500 microM DIDS, 100 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), or 25 microM niflumic acid, suggesting a role for NH(4)(+) action through an apical anion exchanger. mRNA and protein for the apical anion exchangers SLC26A3 [downregulated in adenoma (DRA)] and SLC26A6 [putative anion transporter (PAT1)] were detected in T84 cells by RT-PCR and Northern and Western blots. DRA and PAT1 appear to associate with CFTR in the apical membrane. We conclude that the HCO(3)(-) dependence of apical NH(4)(+) inhibition of secretion is due to the action of NH(4)(+) on an apical anion exchanger.     

Contrasting effects of cPLA2 on epithelial Na+ transport

Activity of the epithelial Na+ channel (ENaC) is the limiting step for discretionary Na+ reabsorption in the cortical collecting duct. Xenopus laevis kidney A6 cells were used to investigate the effects of cytosolic phospholipase A2 (cPLA2) activity on Na+ transport. Application of aristolochic acid, a cPLA2 inhibitor, to the apical membrane of monolayers produced a decrease in apical [3H]arachidonic acid (AA) release and led to an approximate twofold increase in transepithelial Na+ current. Increased current was abolished by the nonmetabolized AA analog 5,8,11,14-eicosatetraynoic acid (ETYA), suggesting that AA, rather than one of its metabolic products, affected current. In single channel studies, ETYA produced a decrease in ENaC open probability. This suggests that cPLA2 is tonically active in A6 cells and that the end effect of liberated AA at the apical membrane is to reduce Na+ transport via actions on ENaC. In contrast, aristolochic acid applied basolaterally inhibited current, and the effect was not reversed by ETYA. Basolateral application of the cyclooxygenase inhibitor ibuprofen also inhibited current. Both effects were reversed by prostaglandin E2 (PGE2). This suggests that cPLA2 activity and free AA, which is metabolized to PGE2, are necessary to support transport. This study supports the fine-tuning of Na+ transport and reabsorption through the regulation of free AA and AA metabolism.     

A quantitative analysis of the interplay of environment,neighborhood, and cell state in 3D spheroids

Cells react to their microenvironment by integrating external stimuli into phenotypic decisions via an intracellular signaling network. To analyze the interplay of environment, local neighborhood, and internal cell state effects on phenotypic variability, we developed an experimental approach that enables multiplexed mass cytometric imaging analysis of up to 240 pooled spheroid microtissues. We quantified the contributions of environment, neighborhood, and intracellular state to marker variability in single cells of the spheroids. A linear model explained on average more than half of the variability of 34 markers across four cell lines and six growth conditions. The contributions of cell‐intrinsic and environmental factors to marker variability are hierarchically interdependent, a finding that we propose has general implications for systems‐level studies of single‐cell phenotypic variability. By the overexpression of 51 signaling protein constructs in subsets of cells, we also identified proteins that have cell‐intrinsic and cell‐extrinsic effects. Our study deconvolves factors influencing cellular phenotype in a 3D tissue and provides a scalable experimental system, analytical principles, and rich multiplexed imaging datasets for future studies.     

Ammonium transport in the colonic crypt cell line, T84: role for Rhesus glycoproteins and NKCC1

Although colonic lumen NH(4)(+) levels are high, 15-44 mM normal range in humans, relatively few studies have addressed the transport mechanisms for NH(4)(+). More extensive studies have elucidated the transport of NH(4)(+) in the kidney collecting duct, which involves a number of transporter processes also present in the distal colon. Similar to NH(4)(+) secretion in the renal collecting duct, we show that the distal colon secretory model, T84 cell line, has the capacity to secrete NH(4)(+) and maintain an apical-to-basolateral NH(4)(+) gradient. NH(4)(+) transport in the secretory direction was supported by basolateral NH(4)(+) loading on NKCC1, Na(+)-K(+)-ATPase, and the NH(4)(+) transporter, RhBG. NH(4)(+) was transported on NKCC1 in T84 cells nearly as well as K(+) as determined by bumetanide-sensitive (86)Rb-uptake. (86)Rb-uptake and ouabain-sensitive current measurement indicated that NH(4)(+) is transported by Na(+)-K(+)-ATPase in these cells to an equal extent as K(+). T84 cells expressed mRNA for the basolateral NH(4)(+) transporter RhBG and the apical NH(4)(+) transporter RhCG. Net NH(4)(+) transport in the secretory direction determined by (14)C-methylammonium (MA) uptake and flux occurred in T84 cells suggesting functional RhG protein activity. The occurrence of NH(4)(+) transport in the secretory direction within a colonic crypt cell model likely serves to minimize net absorption of NH(4)(+) because of surface cell NH(4)(+) absorption. These findings suggest that we rethink the present limited understanding of NH(4)(+) handling by the distal colon as being due solely to passive absorption.     

A Cross-Sectional Study of Water,Sanitation, and Hygiene-Related Risk Factors for Soil-Transmitted Helminth Infection in Urban School- and Preschool-Aged Children in Kibera,Nairobi

Soil-transmitted helminth (STH) infections affect persons living in areas with poor water, sanitation, and hygiene (WASH). Preschool-aged children (PSAC) and school-aged children (SAC) are disproportionately affected by STH infections. We aimed to identify WASH factors associated with STH infection among PSAC and SAC in Kibera, Kenya. In 2012, households containing a PSAC or SAC were randomly selected from those enrolled in the International Emerging Infections Program, a population-based surveillance system. We administered a household questionnaire, conducted environmental assessments for WASH, and tested three stools from each child for STH eggs using the Kato-Katz method. WASH factors were evaluated for associations with STH infection using univariable and multivariable Poisson regression. Any-STH prevalence was 40.8% among 201 PSAC and 40.0% among 475 SAC enrolled. Using the Joint Monitoring Programme water and sanitation classifications, 1.5% of households reported piped water on premises versus 98.5% another improved water source; 1.3% reported improved sanitation facilities, while 81.7% used shared sanitation facilities, 13.9% had unimproved facilities, and 3.1% reported no facilities (open defecation). On univariable analysis, STH infection was significantly associated with a household toilet located off-premises (prevalence ratio (PR) = 1.33; p = 0.047), while always treating water (PR = 0.81; p = 0.04), covering drinking water containers (PR = 0.75; p = 0.02), using clean towels during hand drying (PR = 0.58; p<0.01), having finished household floor material (PR = 0.76; p<0.01), having electricity (PR = 0.70; p<0.01), and increasing household elevation in 10-meter increments (PR = 0.89; p<0.01) were protective against STH infection. On multivariable analysis, usually versus always treating water was associated with increased STH prevalence (adjusted prevalence ratio (aPR) = 1.52; p<0.01), while having finished household floor material (aPR = 0.76; p = 0.03), reported child deworming in the last year (aPR = 0.76; p<0.01), and 10-meter household elevation increases (aPR = 0.89; p<0.01) were protective against infection. The intersection between WASH and STH infection is complex; site-specific WASH interventions should be considered to sustain the gains made by deworming activities.     

2,4-Diaminopyrimidine inhibitors of c-Met kinase bearing benzoxazepine anilines

Elaboration of the SAR around a series of 2,4-diaminopyrimidines led to a number of c-Met inhibitors in which kinase selectivity was modulated by substituents appended on the C4-aminobenzamide ring and the nature of the C2-aminoaryl ring. Further lead optimization of the C2-aminoaryl group led to benzoxazepine analogs whose pharmaceutical properties were modulated by the nature of the substituent on the benzoxazepine nitrogen. Tumor stasis (with partial regressions) were observed when an orally bioavailable analog was evaluated in a GTL-16 tumor xenograft mouse model. Subsequent PK/PD studies suggested that a metabolite contributed to the overall in vivo response.