Nonselective cation transport in native esophageal epithelia

Rabbit esophageal epithelia actively transport Na(+) in a manner similar to that observed in classic electrically tight Na(+)-absorbing epithelia, such as frog skin. However, the nature of the apical entry step is poorly understood. To address this issue, we examined the electrophysiological and biochemical nature of this channel. Western blotting experiments with epithelial Na(+) channel (ENaC) subunit-specific antibodies revealed the presence of all three ENaC subunits in both native and immortalized esophageal epithelial cells. The amino acid sequence of the rabbit alpha-ENaC cloned from native rabbit esophageal epithelia was not significantly different from that of other published alpha-ENaC homologs. To characterize the electrophysiological properties of this native apical channel, we utilized nystatin permeabilization to eliminate the electrical contribution of the basolateral membrane in isolated native epithelia mounted in Ussing-type chambers. We find that the previously described apical Na(+) channel is nonselective for monovalent cations (Li(+), Na(+), and K(+)). Moreover, this channel was not blocked by millimolar concentrations of amiloride. These findings document the presence of a nonselective cation channel in a native Na(+) transporting epithelia, a finding that hereto has been thought to be limited to artificial culture conditions. Moreover, our data are consistent with a potential role of ENaC subunits in the formation of a native nonselective cation channel.     

Interacting domains in the epithelial sodium channel that mediate proteolytic activation

Epithelial Sodium Channel (ENaC) proteolysis at sites in the extracellular loop of the α and γ subunits leads to marked activation. The mechanism of this effect remains debated, as well as the role of the N- and C-terminal fragments of these subunits created by cleavage. We introduced cysteines at sites bracketing upstream and downstream the cleavage regions in α and γ ENaC to examine the role of these fragments in the activated channel. Using thiol modifying reagents, as well as examining the effects of cleavage by exogenous proteases we constructed a functional model that determines the potential interactions of the termini near the cleavage regions. We report that the N-terminal fragments of both α and γ ENaC interact with the channel complex; with interactions between the N-terminal γ and the C-terminal α fragments being the most critical to channel function and activation by exogenous cleavage by subtilisin. Positive charge modification at a.a.135 in the N-terminal fragment of γ exhibited the largest inhibition of channel function. This region was found to interact with the C-terminal α fragment between a.a. 205 and 221; a tract which was previously identified to be the site of subtilisin''s action. These data provide the first evidence for the functional channel rearrangement caused by proteolysis of the α and γ subunit and indicate that the untethered N-terminal fragments of these subunits interact with the channel complex.     

Acute cholesterol-induced anti-natriuretic effects: role of epithelial Na+ channel activity, protein levels, and processing

The epithelial Na(+) channel (ENaC) is modulated by membrane lipid composition. However, the effect of an in vivo change of membrane composition is unknown. We examined the effect of a 70-day enhanced cholesterol diet (ECD) on ENaC and renal Na(+) handling. Rats were fed a standard chow or one supplemented with 1% cholesterol and 0.5% cholic acid (ECD). ECD animals exhibited marked anti-diuresis and anti-natriuresis (40 and 47%), which peaked at 1-3 weeks. Secondary compensation returned urine output and urinary Na(+) excretion to control levels by week 10. During these initial changes, there were no accompanying effects on systolic blood pressure, serum creatinine, or urinary creatinine excretion, indicating that the these effects of ECD preceded those which modify renal filtration and blood pressure. The effects of ECD on ENaC were evaluated by measuring the relative protein content of α, β, and γ subunits. α and γ blots were further examined for subunit cleavage (a process that activates ENaC). No significant changes were observed in α and β levels throughout the study. However, levels of cleaved γ were elevated, suggesting that ENaC was activated. The changes of γ persisted at week 10 and were accompanied by additional subunit fragments, indicating potential changes of γ-cleaving proteases. Enhanced protease activity, and specifically that which could act on the second identified cleavage site in γ, was verified in a newly developed urinary protease assay. These results predict enhanced ENaC activity, an effect that was confirmed in patch clamp experiments of principal cells of split open collecting ducts, where ENaC open probability was increased by 40% in the ECD group. These data demonstrate a complex series of events and a new regulatory paradigm that is initiated by ECD prior to the onset of elevated blood pressure. These events lead to changes of renal Na(+) handling, which occur in part by effects on extracellular γ-ENaC cleavage.     

Bisphosphonates stimulate an endogenous nonselective cation channel in Xenopus oocytes: potential mechanism of action

The mechanisms of action of bisphosphonates (BPs) have been poorly determined. Besides their actions on osteoclasts, these agents exhibit gastrointestinal complications. They have also recently been described as affecting various preparations that express an epithelial Na⁺ channel (ENaC). To understand the effects of BP on ion channels and the ENaC in particular, we used the Xenopus oocyte expression system. Alendronate, and similarly risedronate, two aminobisphosphonates, caused a large stimulation of an endogenous nonselective cation conductance (NSCC). This stimulation averaged 63 ± 12 µS (n = 18) 60 min after the addition of 2 mM alendronate. The effects on the endogenous NSCC were blocked by extracellular acidification to pH 6.4. On the other hand, alendronate caused a small inhibition of ENaC conductance at pH 7.4 and 6.4, but the effects at pH 6.4 were more readily observed in the absence of changes of the endogenous conductance. The effects on membrane capacitance were also markedly different, with a clear decrease at pH 6.4 and no consistent changes at pH 7.4. The effects on the endogenous channel were further augmented by genistein and were inhibited by a tyrosine phosphatase inhibitor, indicating the involvement of the tyrosine kinase pathway. Stimulation of NSCC with BP is expected to cause membrane depolarization and may explain, in part, its mechanisms of action in inhibiting osteoclasts. membrane depolarization; membrane area; drug action     

Role of PKCalpha in feedback regulation of Na(+) transport in an electrically tight epithelium

It has long been known thatNa⁺ channels in electrically tight epithelia are regulatedby homeostatic mechanisms that maintain a steady state and allow newlevels of transport to be sustained in hormonally challenged cells.Little is known about the potential pathways involved in theseprocesses. In addition to short-term effect, recent evidence alsoindicates the involvement of PKC in the long-term regulation of theepithelial Na⁺ channel (ENaC) at the protein level(40). To determine whether stimulation of ENaC involvesfeedback regulation of PKC levels, we utilized Western blot analysis todetermine the distribution of PKC isoforms in polarized A6 epithelia.We found the presence of PKC isoforms in the conventional ( and), novel (, , and ), and atypical (, , and) groups. Steady-state stimulation of Na⁺ transport withaldosterone was accompanied by a specific decrease of PKC proteinlevels in both the cytoplasmic and membrane fractions. Similarly,overnight treatment with an uncharged amiloride analog (CDPC), aprocedure that through feedback regulation causes a stimulation ofNa⁺ transport, also decreased PKC levels. These effectswere additive, indicating separate mechanisms that converge at thelevel of PKC. These effects were not accompanied by changes ofPKC mRNA levels as determined by Northern blot analysis. We proposethat this may represent a novel regulatory feedback mechanism necessary for sustaining an increase of Na⁺ transport.

     

Triangular gold nanoparticles modify shell characteristics and increase antioxidant enzyme activities in the clam Ruditapes decussatus

Context: Nanoparticles may cause adverse environmental effects but there is limited information on their interactions with marine organisms.

Objective: Our aim was to examine the effects of triangular gold nanoparticles (Tr-Au NPs) on the clam, Ruditapes decussatus.

Materials and methods: Clams were exposed to Tr-Au1?=?5?µg/L and Tr-Au2?=?10?µg/L for 2 and 7?days. Effects on shell structure were investigated. Superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST) activities, protein carbonyl levels and malondialdehyde content were used to assess biochemical status.

Results: Transmission electron microscopy (TEM) and electron dispersive X-ray microanalysis (EDX) showed that Tr-Au NPs modified shell structure and morphology. Tr-Au NPs size increased forming aggregate particles. Tr-Au NPs increased SOD, CAT and GST activities in gill and digestive gland in a concentration- and time-dependent manner indicating defence against oxidative stress. Enhanced lipid peroxidation and protein carbonyl levels confirmed oxidative stress.

Conclusion: Tr-Au NPs cause oxidative stress and affect shell structure of clams. These findings may have relevance to other marine species.     

A long isoform of the epithelial sodium channel alpha subunit forms a highly active channel

A long isoform of the human Epithelial Sodium Channel (ENaC) α subunit has been identified, but little data exist regarding the properties or regulation of channels formed by α₇₂₈. The baseline whole cell conductance of oocytes expressing trimeric α₇₂₈βγ channels was 898.1 ± 277.2 and 49.59 ± 13.2 µS in low and high sodium solutions, respectively, and was 11 and 2 fold higher than the conductances of α₆₆₉βγ in same solutions. α₇₂₈βγ channels were also 2 to 5 fold less sensitive to activation by the serine proteases subtilisin and trypsin than α₆₆₉βγ in low and high Na⁺ conditions. The long isoform exhibited lower levels of full length and cleaved protein at the plasma membrane and a rightward shifted sensitivity to inhibition by increases of [Na⁺]_i. Both channels displayed similar single channel conductances of 4 pS, and both were activated to a similar extent by reducing temperature, altogether indicating that activation of baseline conductance of α₇₂₈βγ was likely mediated by enhanced channel activity or open probability. Expression of α₇₂₈ in native kidneys was validated in human urinary exosomes. These data demonstrate that the long isoform of αENaC forms the structural basis of a channel with different activity and regulation, which may not be easily distinguishable in native tissue, but may underlie sodium hyperabsorption and salt sensitive differences in humans.     

Moroccan mitochondrial genetic background suggests prehistoric human migrations across the Gibraltar Strait

Migrations into Africa from the Levant have greatly determined the mitochondrial genetic landscape of North Africa. After analyzing samples from North Morocco to Spain, we show that three fourths of the Moroccan individuals belong to Western Eurasian haplogroups and the frequencies of these are much more similar to those of the Iberian Peninsula than to those of the Middle East. This is particularly true for the mitochondrial haplogroups H1, H3 and V, which experienced a late-glacial expansion from this region, that repopulated much of Central and Northern Europe. Iberian Peninsula was also a source for prehistoric migrations to North Africa.     

Regulation of a cloned epithelial Na+ channel by its beta - and gamma -subunits

Using the Xenopus oocyteexpression system, we examined the mechanisms by which the - and-subunits of an epithelial Na⁺channel (ENaC) regulate -subunit channel activity and the mechanisms by which -subunit truncations cause ENaC activation. Expression of-ENaC alone produced small amiloride-sensitive currents (43 ± 10 nA, n = 7). These currentsincreased >30-fold with the coexpression of - and -ENaC to1,476 ± 254 nA (n = 20).This increase was accompanied by a 3.1- and 2.7-fold increase ofmembrane fluorescence intensity in the animal and vegetal poles of theoocyte, respectively, with use of an antibody directed against the-subunit of ENaC. Truncation of the last 75 amino acids of the-subunit COOH terminus, as found in the original pedigree ofindividuals with Liddle's syndrome, caused a 4.4-fold(n = 17) increase of theamiloride-sensitive currents compared with wild-type -ENaC.This was accompanied by a 35% increase of animal pole membranefluorescence intensity. Injection of a 30-amino acid peptide withsequence identity to the COOH terminus of the human -ENaCsignificantly reduced the amiloride-sensitive currents by 40-50%.These observations suggest a tonic inhibitory role on the channel'sopen probability (P_o) by the COOH terminus of -ENaC. We conclude that the changes of current observed with coexpression of the - and -subunits or those observed with -subunit truncation are likely the result ofchanges of channel density in combination with large changes ofP_o.