ENaC Channels in Oocytes from <i>Xenopus laevis</i> and their Regulation by xShroom1 Protein

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in X. oocytes and is required for the expression of amiloride sensitive sodium channels (ENaC). Oocytes were injected with a, b, and g mENaC and xShroom1 sense or antisense oligonucleotides. We used voltage clamp techniques to study the amiloride-sensitive Na(+) currents (INa((amil))). We observed a marked reduction in INa((amil)) in oocytes co-injected with xShroom1 antisense. Oocytes expressing a DEG mutant â-mENaC subunit (â-S518K) with an open probability of 1 had enhanced INa((amil)) although these currents were also reduced when co-injected with xShroom1 antisense. Addition of low concentration (20 ng/ml) of trypsin which activates the membrane-resident ENaC channels led to a slow increase in INa((amil)) in oocytes with xShroom1 sense but had no effect on the currents in oocytes coinjected with ENaC and xShroom1 antisense. The same results were obtained with higher concentrations of trypsin (2 ug/ml) exposed during 2.5 min. In addition, fluorescence positive staining of plasma membrane in the oocytes expressing a, b and g mENaC and xShroom1 sense were observed but not in oocytes coinjected with ENaC and xShroom1 antisense oligonucleotides. On this basis, we suggest that xShroom1-dependent ENaC inhibition may be through the number of channels inserted in the membrane.Fil: Assef, Yanina Andrea. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ozu, Marcelo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marino, Gabriela Inés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Galizia, Luciano. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kotsias, Basilio Aristides. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

The Variations of Glycolysis and TCA Cycle Intermediate Levels Grown in Iron and Copper Mediums of Trichoderma harzianum

© 2015, Springer Science+Business Media New York.The efficiency of optimal metabolic function by microorganism depends on various parameters, especially essential metal supplementation. In the present study, the effects of iron and copper metals on metabolism were investigated by determination of glycolysis and tricarboxylic acid (TCA) cycle metabolites’ levels with respect to the metal concentrations and incubation period in Trichoderma harzianum. The pyruvate and citrate levels of T. harzianum increased up to 15 mg/L of copper via redirection of carbon flux though glycolysis by suppression of pentose phosphate pathway (PPP). However, the α-ketoglutarate levels decreased at concentration higher than 5 mg/L of copper to overcome damage of oxidative stress. The fumarate levels correlated with the α-ketoglutarate levels because of substrate limitation. Besides, in T. harzianum cells grown in various concentrations of iron-containing medium, the intracellular pyruvate, citrate, and α-ketoglutarate levels showed positive correlation with iron concentration due to modifying of expression of glycolysis and TCA cycle enzymes via a mechanism involving cofactor or allosteric regulation. However, as a result of consuming of prior substrates required for fumarate production, its levels rose up to 10 mg/L

Caffeine intake antagonizes salt sensitive hypertension through improvement of renal sodium handling

High salt intake is a major risk factor for hypertension. Although acute caffeine intake produces moderate diuresis and natriuresis, caffeine increases the blood pressure (BP) through activating sympathetic activity. However, the long-term effects of caffeine on urinary sodium excretion and blood pressure are rarely investigated. Here, we investigated whether chronic caffeine administration antagonizes salt sensitive hypertension by promoting urinary sodium excretion. Dahl salt-sensitive (Dahl-S) rats were fed with high salt diet with or without 0.1% caffeine in drinking water for 15 days. The BP, heart rate and locomotor activity of rats was analyzed and urinary sodium excretion was determined. The renal epithelial Na(+) channel (ENaC) expression and function were measured by in vivo and in vitro experiments. Chronic consumption of caffeine attenuates hypertension induced by high salt without affecting sympathetic nerve activity in Dahl-S rats. The renal α-ENaC expression and ENaC activity of rats decreased after chronic caffeine administration. Caffeine increased phosphorylation of AMPK and decrease α-ENaC expression in cortical collecting duct cells. Inhibiting AMPK abolished the effect of caffeine on α-ENaC. Chronic caffeine intake prevented the development of salt-sensitive hypertension through promoting urinary sodium excretion, which was associated with activation of renal AMPK and inhibition of renal tubular ENaC

Resveratrol Inhibits the Epithelial Sodium Channel via Phopshoinositides and AMP-Activated Protein Kinase in Kidney Collecting Duct Cells

Resveratrol, a naturally occurring phytoalexin, has reported cardioprotective, anti-inflammatory, chemopreventative and antidiabetic properties. Several studies indicate the multiple effects of resveratrol on cellular function are due to its inhibition of class 1A phosphoinositide 3-kinase (PI3K) mediated signaling pathways, but it also activates AMP-activated protein kinase (AMPK). As sodium transport in the kidney via the Epithelial Sodium Channel (ENaC) is highly sensitive to changes in phosphoinositide signaling in the membrane and AMPK, we employed resveratrol to probe the relative effects of phosphatidylinositol species in the plasma membrane and AMPK activity and their impact on ENaC activity in mouse cortical collecting duct (mpkCCD(c14)) cells. Here we demonstrate that resveratrol acutely reduces amiloride-sensitive current in mpkCCD(c14) cells. The time course and dose dependency of this inhibition paralleled depletion of the PI(3,4,5)P(3) reporter (AKT-PH) in live-cell microscopy, indicating the early inhibition is likely mediated by resveratrol's known effects on PI3K activity. Additionally, resveratrol induces a late inhibitory effect (4–24 hours) that appears to be mediated via AMPK activation. Resveratrol treatment induces significant AMPK activation compared with vehicle controls after 4 h, which persists through 16 h. Knockdown of AMPK or treatment with the AMPK inhibitor Compound C reduced the late phase of current reduction but had no effect on the early inhibitory activity of resveratrol. Collectively, these data demonstrate that resveratrol inhibits ENaC activity by a dual effect: an early reduction in activity seen within 5 minutes related to depletion of membrane PIP(3), and a sustained late (4–24 h) effect secondary to activation of AMPK