The Polarized Effect of Intracellular Calcium on the Renal Epithelial Sodium Channel Occurs as a Result of Subcellular Calcium Signaling Domains Maintained by Mitochondria

The renal epithelial sodium channel (ENaC) provides regulated sodium transport in the distal nephron. The effects of intracellular calcium ([Ca²⁺]_i) on this channel are only beginning to be elucidated. It appears from previous studies that the [Ca²⁺]_i increases downstream of ATP administration may have a polarized effect on ENaC, where apical application of ATP and the subsequent [Ca²⁺]_i increase have an inhibitory effect on the channel, whereas basolateral ATP and [Ca²⁺]_i have a stimulatory effect. We asked whether this polarized effect of ATP is, in fact, reflective of a polarized effect of increased [Ca²⁺]_i on ENaC and what underlying mechanism is responsible. We began by performing patch clamp experiments in which ENaC activity was measured during apical or basolateral application of ionomycin to increase [Ca²⁺]_i near the apical or basolateral membrane, respectively. We found that ENaC does indeed respond to increased [Ca²⁺]_i in a polarized fashion, with apical increases being inhibitory and basolateral increases stimulating channel activity. In other epithelial cell types, mitochondria sequester [Ca²⁺]_i, creating [Ca²⁺]_i signaling microdomains within the cell that are dependent on mitochondrial localization. We found that mitochondria localize in bands just beneath the apical and basolateral membranes in two different cortical collecting duct principal cell lines and in cortical collecting duct principal cells in mouse kidney tissue. We found that inhibiting mitochondrial [Ca²⁺]_i uptake destroyed the polarized response of ENaC to [Ca²⁺]_i. Overall, our data suggest that ENaC is regulated by [Ca²⁺]_i in a polarized fashion and that this polarization is maintained by mitochondrial [Ca²⁺]_i sequestration.     

Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition

The stability of Watson–Crick paired RNA/DNA hybrids is important for designing optimal oligonucleotides for ASO (Antisense Oligonucleotide) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 techniques. Previous nearest-neighbour (NN) parameters for predicting hybrid stability in a 1 M NaCl solution, however, may not be applicable for predicting stability at salt concentrations closer to physiological condition (e.g. ∼100 mM Na⁺ or K⁺ in the presence or absence of Mg²⁺). Herein, we report measured thermodynamic parameters of 38 RNA/DNA hybrids at 100 mM NaCl and derive new NN parameters to predict duplex stability. Predicted ΔG°₃₇ and T_m values based on the established NN parameters agreed well with the measured values with 2.9% and 1.1°C deviations, respectively. The new results can also be used to make precise predictions for duplexes formed in 100 mM KCl or 100 mM NaCl in the presence of 1 mM Mg²⁺, which can mimic an intracellular and extracellular salt condition, respectively. Comparisons of the predicted thermodynamic parameters with published data using ASO and CRISPR–Cas9 may allow designing shorter oligonucleotides for these techniques that will diminish the probability of non-specific binding and also improve the efficiency of target gene regulation.     

Differential Responses of CO2 Assimilation,Carbohydrate Allocation and Gene Expression to NaCl Stress in Perennial Ryegrass with Different Salt Tolerance

Little is known about the effects of NaCl stress on perennial ryegrass (Lolium perenne L.) photosynthesis and carbohydrate flux. The objective of this study was to understand the carbohydrate metabolism and identify the gene expression affected by salinity stress. Seventy-four days old seedlings of two perennial ryegrass accessions (salt-sensitive ‘PI 538976’ and salt-tolerant ‘Overdrive’) were subjected to three levels of salinity stress for 5 days. Turf quality in all tissues (leaves, stems and roots) of both grass accessions negatively and significantly correlated with GFS (Glu+Fru+Suc) content, except for ‘Overdrive’ stems. Relative growth rate (RGR) in leaves negatively and significantly correlated with GFS content in ‘Overdrive’ (P<0.01) and ‘PI 538976’ (P<0.05) under salt stress. ‘Overdrive’ had higher CO₂ assimilation and F_v/F_m than ‘PI 538976’. Intercellular CO₂ concentration, however, was higher in ‘PI 538976’ treated with 400 mM NaCl relative to that with 200 mM NaCl. GFS content negatively and significantly correlated with RGR in ‘Overdrive’ and ‘PI 538976’ leaves and in ‘PI 538976’ stems and roots under salt stress. In leaves, carbohydrate allocation negatively and significantly correlated with RGR (r² = 0.83, P<0.01) and turf quality (r² = 0.88, P<0.01) in salt-tolerant ‘Overdrive’, however, the opposite trend for salt-sensitive ‘PI 538976’ (r² = 0.71, P<0.05 for RGR; r² = 0.62, P>0.05 for turf quality). A greater up-regulation in the expression of SPS, SS, SI, 6-SFT gene was observed in ‘Overdrive’ than ‘PI 538976’. A higher level of SPS and SS expression in leaves was found in ‘PI 538976’ relative to ‘Overdrive’. Accumulation of hexoses in roots, stems and leaves can induce a feedback repression to photosynthesis in salt-stressed perennial ryegrass and the salt tolerance may be changed with the carbohydrate allocation in leaves and stems.     

The separation and characterization of two forms ofTorpedo electric organ calelectrin

Two methods for extracting calelectrin, a Ca²⁺-regulated membrane-binding protein from the electric organ ofTorpedo marmorata, have been compared and the more promising one was modified to increase the yield to 7–8 mg · kg⁻¹ wet weight of tissue, that is 4–5-times greater than the original method. The calelectrin so obtained could be resolved into a minor component (designated L-calelectrin) eluted from an anion-exchange column at relatively low ionic strength (100 mM NaCl) and a major component (H-calelectrin) eluted at higher ionic strength (300 mM NaCl). The two forms were also separated by chromatography on a hydrophobic resin. Electrophoresis on cellulose acetate indicated that L-calelectrin had a lower mean isoelectric point that the H-form and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed that under reducing conditions (presence of 5% β-mercaptoethanol) both forms migrated as single species, the L-form having a lower apparent relative molecular mass (M_r 32 000) than the H-form (34 000). Under non-reducing conditions, there was no change in the migration of L-calelectrin but the H-form was resolved into two components of M_r 34 000 and 32 000. The addition of 2 mM Ca²⁺ had no effect on the migration of either form. Both forms were equally recognized by an anti-calelectrin antiserum and were microheterogeneous with respect to their isoelectric points (pH 4.3–5.5) in two-dimensional gel electrophoresis. Physical measurements were carried out on the major H-form. The Stokes radius was estimated to be 3 nm, corresponding to an apparent M_r of 44 000. It was unaffected by changes in ionic strength, pH or Ca²⁺ concentration. Analytical ultracentrifugation gave a sedimentation constant of 2.9 S and an apparent M_r of 36 000. Measurements of circular dichroism indicated that 78% of the molecule was in the α-helix configuration and 22% in random coil. Ca²⁺ had no significant effect on the conformation.     

Adhesion of Annexin 7 Deficient Erythrocytes to Endothelial Cells

Annexin 7 deficiency has previously been shown to foster suicidal death of erythrocytes or eryptosis, which is triggered by increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and characterized by cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface. Eryptosis following increase of [Ca²⁺]_i by Ca²⁺ ionophore ionomycin, osmotic shock or energy depletion was more pronounced in erythrocytes from annexinA7-deficient mice (anxA7^−/−) than in erythrocytes from wild type mice (anxA7^+/+). As phosphatidylserine exposure is considered to mediate adhesion of erythrocytes to the vascular wall, the present study explored adhesion of erythrocytes from anx7^−/− and anx7^+/+-mice following increase of [Ca²⁺]_i by Ca²⁺ ionophore ionomycin (1 µM for 30 min), hyperosmotic shock (addition of 550 mM sucrose for 2 hours) or energy depletion (removal of glucose for 12 hours). Phosphatidylserine exposing erythrocytes were identified by annexin V binding, cell volume estimated from forward scatter in FACS analysis and adhesion to human umbilical vein endothelial cells (HUVEC) utilizing a flow chamber. As a result, ionomycin, sucrose addition and glucose removal all triggered phosphatidylserine-exposure, decreased forward scatter and enhanced adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC), effects significantly more pronounced in anx7^−/− than in anx7^+/+-erythrocytes. Following ischemia, morphological renal injury was significantly higher in anx7^−/− than in anx7^+/+-mice. The present observations demonstrate that enhanced eryptosis of annexin7 deficient cells is paralleled by increased adhesion of erythrocytes to the vascular wall, an effect, which may impact on microcirculation during ischemia.     

Influence of ionic interactions on essential oil and phenolic diterpene composition of Dalmatian sage (Salvia officinalis L.)

The potential of four essential cations (K⁺, Ca²⁺, Mg²⁺ and Fe²⁺) to alleviate salt toxicity was studied in sage (Salvia officinalis L.) plants grown in pots. Two concentrations of the following chloride salts: KCl, CaCl₂, MgCl₂ and FeCl_3, were used together with 100 mM NaCl to study the effects of these nutrients on plant growth, leaf essential oils (EOs) and phenolic diterpenes composition. The sage plants accumulated Na⁺ in their leaves (includers); this has affected secondary metabolites’ biosynthesis. Treatment with 100 mM NaCl slightly decreased borneol and viridiflorol, while increased manool concentrations. Addition of KCl, CaCl₂ and MgCl₂ increased considerably in a dose-dependent manner the oxygen-containing monoterpenes (1.8-cineole, camphor, β-thujone and borneol) in 100 mM NaCl-treated sage. Whereas, the contents of viridiflorol decreased further with the addition of KCl in 100 mM NaCl-treated sage. Our results suggest that the changes in EOs composition were more related to K⁺ and Ca²⁺ availability than to Na⁺ toxicity. Furthermore, treatment with NaCl decreased by 50% carnosic acid (CA), a potent antioxidant, content in the leaves. K⁺ and Ca²⁺ promoted the accumulation of CA and its methoxylated form (MCA) in the leaves. The concentration of CA was positively correlated with leaf K⁺ (r = 0.56, P = 0.01) and Ca²⁺ (r = 0.44, P = 0.05) contents. It appears that different salt applications in combination with NaCl treatments had a profound effect on EOs and phenolic diterpene composition in sage. Therefore, ionic interactions may be carefully considered in the cultivation of this species to get the desired concentrations of these secondary metabolites in leaf extracts.     

Increased cucumber salt tolerance by grafting on pumpkin rootstock and after application of calcium

Self-grafted and pumpkin rootstock-grafted cucumber plants were subjected to the following four treatments: 1) aerated nutrient solution alone (control), 2) nutrient solution with 10 mM Ca(NO₃)₂ (Ca), 3) nutrient solution with 90 mM NaCl (NaCl), and 4) nutrient solution with 90 mM NaCl + 10 mM Ca(NO₃)₂ (NaCl+Ca). The NaCl treatment decreased the plant dry mass and content of Ca²⁺ and K⁺ but increased the Na⁺ content in roots and shoots. Smaller changes were observed in pumpkin rootstock-grafted plants. Supplementary Ca(NO₃)₂ ameliorated the negative effects of NaCl on plant dry mass, relative growth rate (RGR), as well as Ca²⁺, K⁺, and Na⁺ content especially for pumpkin rootstock-grafted plants. Supplementary Ca(NO₃)₂ distinctly stimulated the plasma membrane (PM) H⁺-ATPase activity which supplies the energy to remove excess Na⁺ from the cells. The expressions of gene encoding PM H⁺-ATPases (PMA) and gene encoding a PM Na⁺/H⁺ antiporter (SOS1) were up-regulated when Ca(NO₃)₂ was applied. The pumpkin rootstock-grafted plants had higher PM H⁺-ATPase activity as well as higher PMA and SOS1 expressions than the self-grafted plants under NaCl + Ca treatment. Therefore, the addition of Ca²⁺ in combination with pumpkin rootstock grafting is a powerful way to increase cucumber salt tolerance.     

Effects of Salt Stress on Photosynthetic Pigments and Activity of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in <Emphasis Type="Italic">Kalidium foliatum</Emphasis>

The effects of NaCl and Na₂SO₄ on photosynthetic pigments, malondialdehyde (MDA), Rubisco activity and superoxide dismutase (SOD) activity were investigated in Kalidium foliatum (Pall.) Moq., which is distributed in the saline soil of Hetao irrigation area in Inner Mongolia China. The K. foliatum plants were treated with NaCl (0, 100, 250, 400 and 500 mM), Na₂SO₄ (0, 100, 250, 400 and 500 mM) and NaCl + Na₂SO₄ (1: 1, v/v) (0, 100, 250, 400 and 500 mM of Na⁺ concentration, 0, 50, 125, 200 and 250 mM of Cl^– and SO ₄ ^2– concentration) for 10 days. Content of chlorophylls and carotenoids were significantly higher than control at increasing NaCl and Na₂SO₄ concentration, in contrast, were significantly reduced by higher concentration of NaCl + Na₂SO₄. Rubisco activity reduced steadily at 100 and 250 mM NaCl, while increased at 400 and 500 mM NaCl. Rubisco activity was significantly higher than control at 100 mM Na₂SO₄, and was no more change under NaCl + Na₂SO₄ treatment. The SOD activity increased with increasing NaCl and Na₂SO₄, and increased at moderate NaCl + Na₂SO₄ treatment. MDA content was lower than control at 250 mM salt concentration. On the basis of the data obtained, K. foliatum showed resistance to salt such as Na⁺, Cl^–and SO ₄ ^2– , Rubisco activity in K. foliatum might be more sensitive to salt.     

Effects of Salinity on Water Transport of Excised Maize (Zea mays L.) Roots

The root pressure probe was used to determine the effects of salinity on the hydraulic properties of primary roots of maize (Zea mays L. cv Halamish). Maize seedlings were grown in nutrient solutions modified by additions of NaCl and/or extra CaCl₂ so that the seedlings received one of four treatments: Control, plus 100 millimolar NaCl, plus 10 millimolar CaCl₂, plus 100 millimolar NaCl plus 10 millimolar CaCl₂. The hydraulic conductivities (Lp_r) of primary root segments were determined by applying gradients of hydrostatic and osmotic pressure across the root cylinder. Exosmotic hydrostatic Lp_r for the different treatments were 2.8, 1.7, 2.8, and 3.4·10⁻⁷ meters per second per megapascals and the endosmotic hydrostatic Lp_r were 2.4, 1.5, 2.7, and 2.3·10⁻⁷ meters per second per megapascals, respectively. Exosmotic Lp_r of the osmotic experiments were 0.55, 0.38, 0.68, and 0.60·10⁻⁷ meters per second per megapascals and the endosmotic Lp_r were 0.53, 0.21, 0.56, and 0.54·10⁻⁷ meters per second per megapascals, respectively. The osmotic Lp_r was significantly smaller (4-5 times) than hydrostatic Lp_r. However, both hydrostatic and osmotic Lp_r experiments showed that salinization of the growth media at regular (0.5 millimolar) calcium levels decreased the Lp_r significantly (30-60%). Addition of extra calcium (10 millimolar) to the salinized media caused ameliorative effects on Lp_r. The low Lp_r values may partially explain the reduction in root growth rates caused by salinity. High calcium levels in the salinized media increased the relative availability of water needed for growth. The mean reflection coefficients of the roots using NaCl were between 0.64 and 0.73 and were not significantly different for the different treatments. The mean values of the root permeability coefficients to NaCl of the different treatments were between 2.2 and 3.5·10⁻⁹ meters per second and were significantly different only in one of four treatments. Cutting the roots successively from the tip and measuring the changes in the hydraulic resistance of the root as well as staining of root cross-sections obtained at various distances from the root tip revealed that salinized roots had mature xylem elements closer to the tip (5-10 millimeters) compared with the controls (30 millimeters). Our results demonstrate that salinity has adverse effects on water transport and that extra calcium can, in part, compensate for these effects.     

Interactive effects of Ca2+ and NaCl salinity on the growth of two tomato genotypes differing in Ca2+ use efficiency

Two tomato (Lycopersicon esculentum Mill.) lines differing in Ca²⁺ use efficiency (Ca²⁺ use efficient line 113 and Ca²⁺ use inefficient line 67) were subjected to salinity treatments in two separate experiments to determine whether they differed in salt tolerance. In experiment I, three NaCl and two CaCl₂ treatments were imposed. The Na⁺ concentrations were 1.1, 100 and 150 mM and the Ca²⁺ concentrations were either 1.51 or 10 mM. In experiment II, one NaCl and three Ca²⁺ treatments (as CaCl₂ or CaSO₄) were imposed. The treatments consisted of 150 mM NaCl at either 1.51 mM CaCl₂, 10 mM CaCl₂, or 10 mM CaSO₄. Response to treatments was determined by analysis of growth parameters (shoot and root dry weights, plant height, and root length). Shoot and root dry weight, and root length were depressed as salinity increased in plants lacking additional Ca²⁺. No significant differences in salt tolerance were detected between the two tomato lines after 24 d of salinity treatment. An important finding of this study was that root growth and length appeared to be more sensitive to the effect of CaCI₂ treatment alone and to the effects of CaCl₂ × NaCl treatments. This suggests that over the long term, both root growth and root length may be more sensitive indicators of salinity effects than shoots. Supplemental CaCl₂ had no ameliorative effect on NaCl stress in shoot growth. The inability of Ca²⁺ to counter Cl⁻ entry or toxicity may account for the lack of amelioration. Additional Ca²⁺ as CaSO₄ improved shoot growth of plants exposed to 150 mM NaCl. In contrast, root growth and length were improved by 10 mM Ca²⁺ as either CaCl₂ or CaSO₄.     

The fine structure of euchromatin and centromeric heterochromatin in Tenebrio molitor chromosomes

The fine structure of constitutive heterochromatin and euchromatin was compared in electron microscope whole-mount preparations of Tenebrio molitor (Insecta, Coleoptera) spermatocyte nuclei. Tenebrio molitor pachytene chromosomes display extended segments of centromeric heterochromatin and thus are especially suitable for this purpose. When nuclei were incubated in solutions containing different concentrations of NaCl or of MgCl₂, two levels of chromatin fine structures were observed in the euchromatic segments: nucleosome fibers (0.1 mM–20 mM NaCl) and supranucleosomal fibers with 28 nm in diameter (40 mM–100 mM NaCl, 0.2 mM–1.0 mM MgCl₂). The fine structure in the heterochromatic segments was the same as that in the euchromatic segments in all NaCl concentrations and in MgCl₂ concentrations up to 0.4 mM. In higher MgCl₂ concentrations the heterochromatin remained more compact than the euchromatin and consisted of 37-nm-thick fibers in 0.6 mM MgCl₂ and of 65-nm-thick fibers in 1.0 mM MgCl₂. After the 37-nm and the 65-nm fibers had been dispersed in Mg²⁺-free solutions they could be recondensed by incubation in 0.6 mM and 1.0 mM MgCl₂, respectively. It is concluded that a Mg²⁺-sensitive component of the heterochromatin is responsible for the folding of the nucleosome chain to heterochromatin-specific supranucleosomal structures.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

Effects of metalloendoproteinase inhibitors on secretion and intracellular free calcium in bovine adrenal chromaffin cells

The possible role of metalloendoproteinase in stimulus-secretion coupling in adrenal chromaffin cells was examined using the metalloendoproteinase inhibitors 1,10-phenanthroline and carbobenzoxy-Gly-Phe-NH₂. Catecholamine release elicited by nicotine or by depolarisation with 55 mM K⁺ was almost completely abolished by 0.5 mM 1,10-phenanthroline. Carbobenzoxy-Gly-Phe-NH₂ (2.5 mM) inhibited catecholamine release in response to nicotine but enhanced that due to 55 mM K⁺. The rise in intracellular free calcium, [Ca²⁺]_i, in response to either nicotine or 55 mM was inhibited by about 50% by both inhibitors. One site of action of metalloendoproteinase inhibitors may, therefore, be at the level of the regulation of [Ca²⁺]_i. Catecholamine release and the rise in [Ca²⁺]_i elicited by the calcium ionophore ionomycin were not reduced by the inhibitors. These results show that metalloendoproteinase inhibitors have complex effects on chromaffin cells including effects on the regulation of [Ca²⁺]_i but do not inhibit calcium-activated exocytosis itself.     

Modulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa

Background

The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na⁺-K⁺-ATPase.

Methods

Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na⁺-K⁺-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.

Results

The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α₁Na⁺-K⁺-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.

Conclusions

These results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.     

Identification of Extracellular Domain Residues Required for Epithelial Na+ Channel Activation by Acidic pH

A growing body of evidence suggests that the extracellular domain of the epithelial Na⁺ channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na⁺ self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γ_E455 is conserved in βENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with β_E446A generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na⁺ self-inhibition. Together, the data identify eight extracellular domain residues in human β- and γENaC that are required for regulation by acidic pH.     

Loop dependence of the stability and dynamics of nucleic acid hairpins

Hairpin loops are critical to the formation of nucleic acid secondary structure, and to their function. Previous studies revealed a steep dependence of single-stranded DNA (ssDNA) hairpin stability with length of the loop (L) as ~L^{8.5 ± 0.5}, in 100 mM NaCl, which was attributed to intraloop stacking interactions. In this article, the loop-size dependence of RNA hairpin stabilities and their folding/unfolding kinetics were monitored with laser temperature-jump spectroscopy. Our results suggest that similar mechanisms stabilize small ssDNA and RNA loops, and show that salt contributes significantly to the dependence of hairpin stability on loop size. In 2.5 mM MgCl₂, the stabilities of both ssDNA and RNA hairpins scale as ~L^{4 ± 0.5}, indicating that the intraloop interactions are weaker in the presence of Mg²⁺. Interestingly, the folding times for ssDNA hairpins (in 100 mM NaCl) and RNA hairpins (in 2.5 mM MgCl₂) are similar despite differences in the salt conditions and the stem sequence, and increase similarly with loop size, ~L^{2.2 ± 0.5} and ~L^{2.6 ± 0.5}, respectively. These results suggest that hairpins with small loops may be specifically stabilized by interactions of the Na⁺ ions with the loops. The results also reinforce the idea that folding times are dominated by an entropic search for the correct nucleating conformation.     

Salt tolerance of a cash crop halophyte Suaeda fruticosa: biochemical responses to salt and exogenous chemical treatments

Suaeda fruticosa Forssk is a leaf succulent obligate halophyte that produces numerous seeds under saline conditions. Seeds are a good source of high quality edible oil and leaves are capable of removing substantial amount of salt from the saline soil besides many other economic usages. Little is known about the biochemical basis of salt tolerance in this species. We studied some biochemical responses of S. fruticosa to different exogenous treatments under non-saline (0 mM), moderate (300 mM) or high (600 mM) NaCl levels. Eight-week-old seedlings were sprayed twice a week with distilled water, hydrogen peroxide (H₂O₂, 100 μM), glycine betaine (GB, 10 mM), or ascorbic acid (AsA, 20 mM) for 30 days. At moderate (300 mM) NaCl, leaf Na⁺, Ca²⁺ and osmolality increased, along with unchanged ROS and antioxidant enzyme activities, possibly causing a better plant growth. Plants grew slowly at 600 mM NaCl to avoid leaf Na⁺ buildup relative to those at 300 mM NaCl. Exogenous application of distilled water and H₂O₂ improved ROS scavenging mechanisms, although growth was unaffected. ASA and GB alleviated salt-induced growth inhibition at 600 mM NaCl through enhancing the antioxidant defense system and osmotic and ion homeostasis, respectively.     

Differences in the regulation of RyR2 from human,sheep, and rat by Ca2+ and Mg2+ in the cytoplasm and in the lumen of the sarcoplasmic reticulum

Regulation of the cardiac ryanodine receptor (RyR2) by intracellular Ca²⁺ and Mg²⁺ plays a key role in determining cardiac contraction and rhythmicity, but their role in regulating the human RyR2 remains poorly defined. The Ca²⁺- and Mg²⁺-dependent regulation of human RyR2 was recorded in artificial lipid bilayers in the presence of 2 mM ATP and compared with that in two commonly used animal models for RyR2 function (rat and sheep). Human RyR2 displayed cytoplasmic Ca²⁺ activation (K_a = 4 µM) and inhibition by cytoplasmic Mg²⁺ (K_i = 10 µM at 100 nM Ca²⁺) that was similar to RyR2 from rat and sheep obtained under the same experimental conditions. However, in the presence of 0.1 mM Ca²⁺, RyR2s from human were 3.5-fold less sensitive to cytoplasmic Mg²⁺ inhibition than those from sheep and rat. The K_a values for luminal Ca²⁺ activation were similar in the three species (35 µM for human, 12 µM for sheep, and 10 µM for rat). From the relationship between open probability and luminal [Ca²⁺], the peak open probability for the human RyR2 was approximately the same as that for sheep, and both were ∼10-fold greater than that for rat RyR2. Human RyR2 also showed the same sensitivity to luminal Mg²⁺ as that from sheep, whereas rat RyR2 was 10-fold more sensitive. In all species, modulation of RyR2 gating by luminal Ca²⁺ and Mg²⁺ only occurred when cytoplasmic [Ca²⁺] was <3 µM. The activation response of RyR2 to luminal and cytoplasmic Ca²⁺ was strongly dependent on the Mg²⁺ concentration. Addition of physiological levels (1 mM) of Mg²⁺ raised the K_a for cytoplasmic Ca²⁺ to 30 µM (human and sheep) or 90 µM (rat) and raised the K_a for luminal Ca²⁺ to ∼1 mM in all species. This is the first report of the regulation by Ca²⁺ and Mg²⁺ of native RyR2 receptor activity from healthy human hearts.     

Properties and nature of (Na+ + Mg2+)-dependent adenosine triphosphatase in the gills of the eel, angvilla anguilla (L.)

The specific activity of (Na⁺ + Mg²⁺)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase in both cases.(Na⁺ + Mg²⁺)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI₂, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea.It is concluded that the (Na⁺ + Mg²⁺)-dependent ATPase activity of gills arises from the presence of a (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase.     

Involvement of NADPH-dependent and cAMP-PKA sensitive H+ channels in the chorda tympani nerve responses to strong acids

To investigate if chorda tympani (CT) taste nerve responses to strong (HCl) and weak (CO(2) and acetic acid) acidic stimuli are dependent upon NADPH oxidase-linked and cAMP-sensitive proton conductances in taste cell membranes, CT responses were monitored in rats, wild-type (WT) mice, and gp91(phox) knockout (KO) mice in the absence and presence of blockers (Zn(2+) and diethyl pyrocarbonate [DEPC]) or activators (8-(4-chlorophenylthio)-cAMP; 8-CPT-cAMP) of proton channels and activators of the NADPH oxidase enzyme (phorbol 12-myristate 13-acetate [PMA], H(2)O(2), and nitrazepam). Zn(2+) and DEPC inhibited and 8-CPT-cAMP, PMA, H(2)O(2), and nitrazepam enhanced the tonic CT responses to HCl without altering responses to CO(2) and acetic acid. In KO mice, the tonic HCl CT response was reduced by 64% relative to WT mice. The residual CT response was insensitive to H(2)O(2) but was blocked by Zn(2+). Its magnitude was further enhanced by 8-CPT-cAMP treatment, and the enhancement was blocked by 8-CPT-adenosine-3'-5'-cyclic monophospho-rothioate, a protein kinase A (PKA) inhibitor. Under voltage-clamp conditions, before cAMP treatment, rat tonic HCl CT responses demonstrated voltage-dependence only at ±90 mV, suggesting the presence of H(+) channels with voltage-dependent conductances. After cAMP treatment, the tonic HCl CT response had a quasi-linear dependence on voltage, suggesting that the cAMP-dependent part of the HCl CT response has a quasi-linear voltage dependence between +60 and -60 mV, only becoming sigmoidal when approaching +90 and -90 mV. The results suggest that CT responses to HCl involve 2 proton entry pathways, an NADPH oxidase-dependent proton channel, and a cAMP-PKA sensitive proton channel.