Alterations In Na Research Articles

Phenotypic plasticity in response to dietary salt stress: Na+ and K+ transport by the gut ofDrosophila melanogasterlarvae

Drosophila provides a useful model system for studies of the mechanisms involved in regulation of internal ion levels in response to variations in dietary salt load. This study assessed whether alterations in Na(+) and K(+) transport by the gut of larval D. melanogaster reared on salt-rich diets contribute to haemolymph ionoregulation. Na(+) and K(+) fluxes across the isolated guts of third instar larvae reared on control or salt-rich diets were measured using the scanning ion-selective electrode technique (SIET). K(+) absorption across the anterior portion of the posterior midgut of larvae reared on diet in which the concentration of KCl was increased 0.4 mol l(-1) above that in the control diet was reduced eightfold relative to the same gut segment of larvae reared on the control diet. There was also an increase in the magnitude and extent of K(+) secretion across the posterior half of the posterior midgut. Na(+) was absorbed across the ileum of larvae reared on the control diet, but was secreted across the ileum of larvae reared on diet in which the concentration of NaCl was increased 0.4 mol l(-1) above that in the control diet. There was also a small reduction in the extent of Na(+) absorption across the middle midgut of larvae reared on the NaCl-rich diet. The results indicate considerable phenotypic plasticity with respect to K(+) and Na(+) transport by the gut epithelia of larval D. melanogaster. SIET measurements of K(+) and Na(+) fluxes along the length of the gut show that ion transport mechanisms of the gut are reconfigured during salt stress so that there are reductions in K(+) and Na(+) absorption and increases in K(+) and Na(+) secretion. Together with previously described changes in salt secretion by the Malpighian tubules, these changes contribute to haemolymph ionoregulation.

NaV1.8, but not NaV1.9, is upregulated in the inflamed dental pulp tissue of human primary teeth

To investigate alterations in Na(V) 1.8 and Na(V) 1.9 expression within inflamed dental pulp tissue of human primary teeth. Dental pulp tissue obtained from both normal and inflamed pulps in primary teeth as well as pulps from normal and inflamed permanent teeth was used. The quantity of Na(V) 1.8 and Na(V) 1.9 expression in the dental pulp tissue was investigated using Western blot analysis. General neuron marker (PGP9.5) was used to quantify for neural density, and an increase in metalloproteinase-9 was used to indicate pulpal inflammation in inflamed teeth. Statistically significant differences for each determined parameter between normal and inflamed teeth of both primary and permanent teeth were tested using the Mann-Whitney rank sum test. There was no significant difference in neural density of normal and inflamed dental pulp tissue, although degrees of inflammation were increased in the inflamed dental pulp of both permanent and primary teeth (P < 0.05). Na(V) 1.8 and Na(V) 1.9 expression in inflamed pulps of permanent teeth increased significantly compared with normal permanent teeth (P < 0.05). However, only Na(V) 1.8 expression was increased significantly in the inflamed dental pulp of primary teeth (P < 0.05). Na(V) 1.8 alone may be the therapeutic target for treatment of painful pulpitis in primary teeth.

Beneficial Effects of PKF275-055, a Novel, Selective, Orally Bioavailable, Long-Acting Dipeptidyl Peptidase IV Inhibitor in Streptozotocin-Induced Diabetic Peripheral Neuropathy

1-[(2-adamantyl)amino]acetyl-2-cyano-(S)-pyrrolidine, monohydrochloride (PKF275-055), a vildagliptin analog, is a novel, selective, potent, orally bioavailable, and long-acting dipeptidyl peptidase IV inhibitor. We studied the effect of PKF275-055 administration on the prevention, protection, and treatment of diabetic neuropathy in the streptozotocin-induced diabetic rat. PKF275-055 improved body and muscle weight. Oral glucose tolerance tests showed a marked improvement in glucose metabolism under all treatment schedules. When tested in prevention and protection experiments, PKF275-055 completely averted the decrease of Na⁺/K⁺-ATPase activity and partially counteracted the nerve conduction velocity (NCV) deficit observed in untreated diabetic rats but had no effects on abnormal mechanical and thermal sensitivity. When used in a therapeutic setting, PKF275-055 induced a significant correction in the alteration in Na⁺,K⁺-ATPase activity and NCV present in untreated diabetics. Diabetic rats developed mechanical hyperalgesia within 2 weeks after streptozotocin injection and exhibited significantly longer thermal response latencies. It is noteworthy that PKF275-055 treatment restored mechanical sensitivity thresholds by approximately 50% (p < 0.01) and progressively improved the alteration in thermal responsiveness. In conclusion, PKF275-055 showed an anabolic effect, improved oral glucose tolerance, and counteracted the alterations in Na⁺,K⁺-ATPase activity, NCV, and nociceptive thresholds in diabetic rats. The present data support a potential therapeutic effect of PKF275-055 in the treatment of rodent diabetic neuropathy.

Na+,K+-ATPase activity is increased in rats subjected to chronic administration of ketamine.

De Luca RD, Fraga DB, Ghedim FV, Kolling J, Ferreira AGK, Cunha AA, Wyse ATS, Zugno AI. Na+,K+-ATPase activity is increased in rats subjected to chronic administration of ketamine.Objective: Schizophrenia is a devastating psychiatric disorder. Symptoms of schizophrenia can be divided into positive, negative and cognitive, and the physiopathology is still been unknown. Na+,K+-ATPase is a protein in its role as a maintainer of fluid balance in all mammals and alterations in this enzyme could cause brain abnormalities. The aim of our study was to investigate the activity of this enzyme in rats submitted to chronic administration of ketamine.Methods: Adult male Wistar rats were submitted to sub-anaesthetic doses of the 25 mg/kg ketamine by seven consecutive days and the Na+,K+-ATPase activity was analysed in prefrontal and hippocampus of rats.Results: We observed an increase in Na+,K+-ATPase activity in prefrontal cortex administration of 25 mg/kg ketamine. However, ketamine has no effect in hippocampus.Conclusion: This evidence indicates that the alteration in Na+,K+-ATPase may be related with glutamatergic system and consequently could be related to the development of schizophrenia.

Cystic fibrosis: an-ion transport issue?

Cystic fibrosis can manifest in many organs, but infection and inflammation of the airways cause the most severe morbidity and mortality. Early studies suggested that increased epithelial Na+ reabsorption may be a key initiating event in cystic fibrosis lung disease. However, the importance of Na+ hyperabsorption and the consequent depletion of pericellular airway liquid in cystic fibrosis lung disease has been called into question by a recent study by Jeng-Haur Chen et al.. Newborn pigs lacking cystic fibrosis transmembrane conductance regulator (CFTR−/− pigs) developed lung pathology reminiscent of human cystic fibrosis. Although epithelia from these pigs had reduced Cl− and HCO3− transport, there were no alterations in Na+ or liquid absorption, suggesting that defective anion transport is responsible for cystic fibrosis lung disease. Here three experts comment on how these findings might affect our understanding of cystic fibrosis lung pathogenesis and strategies for cystic fibrosis therapy.

Signaling through the Na/K-ATPase: implications for cardiac fibrosis

the cardiotonic effects of digitalis have been exploited in clinical practice for many years. However, while the existence of endogenous digitalis-like substances (also called cardiotonic steroids) has been accepted for some time, their exact chemical nature has been only recently demonstrated ([1][

Renal aging in WKY rats: Changes in Na +,K +-ATPase function and oxidative stress

It has been suggested that alterations in Na +,K +-ATPase mediate the development of several aging-related pathologies, such as hypertension and diabetes. Thus, we evaluated Na +,K +-ATPase function and H 2O 2 production in the renal cortex and medulla of Wistar Kyoto (WKY) rats at 13, 52 and 91 weeks of age. Creatinine clearance, proteinuria, urinary excretion of Na + and K + and fractional excretion of Na + were also determined. The results show that at 91 weeks old WKY rats had increased creatinine clearance and did not have proteinuria. Despite aging having had no effect on urinary Na + excretion, urinary K + excretion was increased and fractional Na + excretion was decreased with age. In renal proximal tubules and isolated renal cortical cells, 91 week old rats had decreased Na +,K +-ATPase activity when compared to 13 and 52 week old rats. In renal medulla, 91 week old rats had increased Na +,K +-ATPase activity, paralleled by an increase in protein expression of α 1-subunit of Na +,K +-ATPase. In addition, renal H 2O 2 production increased with age and at 91 weeks of age renal medulla H 2O 2 production was significantly higher than renal cortex production. The present work demonstrates that although at 91 weeks of age WKY rats were able to maintain Na + homeostasis, aging was accompanied by alterations in renal Na +,K +-ATPase function. The observed increase in oxidative stress may account, in part, for the observed changes. Possibly, altered Na +,K +-ATPase renal function may precede the development of age-related pathologies and loss of renal function.

Extracellular Allosteric Regulatory Subdomain within the γ Subunit of the Epithelial Na+ Channel

The activity of the epithelial Na(+) channel (ENaC) is modulated by Na(+) self-inhibition, a down-regulation of the open probability of ENaC by extracellular Na(+). A His residue within the extracellular domain of gammaENaC (gammaHis(239)) was found to have a critical role in Na(+) self-inhibition. We investigated the functional roles of residues in the vicinity of this His by mutagenesis and analyses of Na(+) self-inhibition responses in Xenopus oocytes. Significant changes in the speed and magnitude of Na(+) self-inhibition were observed in 16 of the 47 mutants analyzed. These 16 mutants were distributed within a 22-residue tract. We further characterized this scanned region by examining the accessibility of introduced Cys residues to the sulfhydryl reagent MTSET. External MTSET irreversibly increased or decreased currents in 13 of 47 mutants. The distribution patterns of the residues where substitutions significantly altered Na(+) self-inhibition or/and conferred sensitivity to MTSET were consistent with the existence of two helices within this region. In addition, single channel recordings of the gammaH239F mutant showed that, in the absence of Na(+) self-inhibition and with an increased open probability, ENaCs still undergo transitions between open and closed states. We conclude that gammaHis(239) functions within an extracellular allosteric regulatory subdomain of the gamma subunit that has an important role in conferring the response of the channel to external Na(+).

Alterations of Na +/K +-ATPase function in caveolin-1 knockout cardiac fibroblasts

Recent studies have demonstrated that the Na +/K +-ATPase is not only an ion pump, but also a membrane receptor that confers the ligand-like effects of cardiotonic steroids (CTS) such as ouabain on protein kinases and cell growth. Because CTS have been implicated in cardiac fibrosis, this study examined the role of caveolae in the regulation of Na +/K +-ATPase function and CTS signaling in cardiac fibroblasts. In cardiac fibroblasts prepared from wild-type and caveolin-1 knockout [Cav-1(−/−)] mice, we found that the absence of caveolin-1 did not affect total cellular amount or surface expression of Na +/K +-ATPase α1 subunit. However, it did increase ouabain-sensitive 86Rb + uptake. While knockout of caveolin-1 increased basal activities of Src and ERK1/2, it abolished the activation of these kinases induced by ouabain but not angiotensin II. Finally, ouabain stimulated collagen synthesis and cell proliferation in wild type but not Cav-1(−/−) cardiac fibroblasts. Thus, we conclude that caveolae are important for regulating both pumping and signal transducing functions of Na +/K +-ATPase. While depletion of caveolae increases the pumping function of Na +/K +-ATPase, it suppresses CTS-induced signal transduction, growth, and collagen production in cardiac fibroblasts.

Role of Electrolytes and Glucose in the Insulin-Induced Electrochemical Effect in Sheep Pleura

Insulin induces electrochemical alterations in sheep visceral and parietal pleura, an effect abolished by the Na (+)-channel blocker amiloride and the Na (+)-K (+) pump inhibitor ouabain. The aim of this study was to further investigate the role of different electrolytes and glucose in these electrochemical changes. Sheep pleural specimens were mounted in Ussing chambers. Insulin (10 (-7)M) was added mesothelially in Na (+), K (+), Ca (2+)-free, low H (+) and glucose solutions. In other experiments, specimens were pretreated with K (+) and Ca (2+)-free Krebs solutions. Trans-mesothelial Resistance was determined. Insulin did not increase Trans-mesothelial Resistance of visceral and parietal pleura in K (+)-free (p=0.008 and p=0.028 respectively), Ca (2+)-free (p=0.006 and p=0.012 respectively) and low glucose (p=0.009 and p=0.03 respectively) solutions. This effect was totally inhibited in Na (+)-free solutions or in specimens pretreated with Ca (2+) -free Krebs solution and partially inhibited, when low H (+) solutions were used (p=0.042 for visceral and p=0.045 for parietal). Insulin-induced electrochemical changes in sheep pleura are mainly associated with alterations in Na (+) and Ca (2+) concentrations. Since amiloride and ouabain abolish these electrochemical changes, it may be suggested that insulin could influence the pleural fluid recycling, mainly via the Na (+) transportation system, irrespective of the glucose content.

Inhibition of mammal Na(+)K(+)-ATPase by diterpenes extracted from the Brazilian brown alga Dictyota cervicornis.

The dolastane diterpenes 4-acetoxy-9,14-dihydroxydolast-1(15),7-diene (1) and 4,7-diacetoxy-14-hydroxydolast-1(15),8-diene (2) were isolated from specimens of the alga Dictyota cervicornis collected from the Rio de Janeiro coast, Brazil. Chemical structures of the diterpenes were assigned by 1D and 2D NMR spectral data for the first time. Both substances inhibited Na(+)K(+)-ATPase preparations from guinea-pig brain or kidney, with the same inhibitory potency towards enzyme isoforms. The maximal inhibition obtained for 1 was 40% at a concentration of 0.5 mm in the incubation mixture, while it reached 80% for compound 2 at this concentration. Ouabain insensitive ATPases were inhibited by 1, but not by 2. Data comparing the inhibitory potency of these compounds with that of ouabain and oleic acid suggest a higher degree of selectivity of 2 towards the Na(+)K(+)-pump. Cardiac glycosides such as ouabain are used classically in the treatment of heart failure, but alterations of Na(+)K(+)-pump activity are also involved in several other diseases. Therefore, the study of compounds interfering with this pump activity is gaining further importance.

Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide (KCN)/0.1 mM iodoacetic acid (IAA), and membrane transport function and cell viability were evaluated by measuring Na(+)-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in Na(+)-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited Na(+)-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a Na(+) pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in Na(+)-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers (dimethylthiourea and thiourea), and amino acids (glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase A(2) (cPLA(2)). The ATP depletion-dependent arachidonic acid release was inhibited by cPLA(2) specific inhibitor AACOCF(3). ATP depletion-induced alterations in Na(+)-dependent phosphate uptake and cell viability were prevented by AACOCF(3). Inhibition of Na(+)-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and cPLA(2) activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.

Claudin-8 and -27 tight junction proteins in puffer fish Tetraodon nigroviridis acclimated to freshwater and seawater

Genes encoding for claudin-8 and -27 tight junction proteins in the euryhaline puffer fish (Tetraodon nigroviridis) were identified using its recently sequenced genome. Phylogenetic analysis indicated that multiple genes encoding for claudin-8 proteins (designated Tncldn8a, Tncldn8b, Tncldn8c and Tncldn8d) arose by tandem gene duplication. In contrast, both tandem and whole genome duplication events appear to have generated genes encoding for claudin-27 proteins (designated Tncldn27a, Tncldn27b, Tncldn27c and Tncldn27d). Tncldn8 and Tncldn27 mRNA were widely distributed in Tetraodon, suggesting involvement in various physiological processes. All Tncldn8 and Tncldn27 genes were expressed in gill and skin tissue (i.e., epithelia exposed directly to the external environment). A potential role for claudin-8 and -27 proteins in the regulation of hydromineral balance in Tetraodon was investigated by examining alterations in mRNA abundance in select ionoregulatory tissue of fish acclimated to freshwater (FW) and seawater (SW). In FW or SW, Tetraodon exhibited alterations in Na(+)-K(+)-ATPase activity (a correlate of transcellular transport) typical of a euryhaline teleost fish. Simultaneously, tissue and gene specific alterations in Tncldn8 and Tncldn27 transcript abundance occurred. These data provide some insight into the duplication history of cldn8 and cldn27 genes in fishes and suggest a possible role for claudin-8 and -27 proteins in the osmoregulatory strategies of euryhaline teleosts.

Nav1.4 Deregulation in Dystrophic Skeletal Muscle Leads to Na+ Overload and Enhanced Cell Death

Duchenne muscular dystrophy (DMD) is a hereditary degenerative disease manifested by the absence of dystrophin, a structural, cytoskeletal protein, leading to muscle degeneration and early death through respiratory and cardiac muscle failure. Whereas the rise of cytosolic Ca2+ concentrations in muscles of mdx mouse, an animal model of DMD, has been extensively documented, little is known about the mechanisms causing alterations in Na+ concentrations. Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Nav1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na+ concentrations found in muscle from mdx mice. The absence of dystrophin modifies the expression level and gating properties of Nav1.4, leading to an increased Na+ concentration under the sarcolemma. Moreover, the distribution of Nav1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin α-1, thus suggesting that syntrophin is an important linker between dystrophin and Nav1.4. Additionally, we show that these modifications of Nav1.4 gating properties and increased Na+ concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na+ overload can be reversed by 3 nM tetrodotoxin, a specific Nav1.4 blocker.

The peroxynitrite donor 3-morpholinosydnonimine induces reversible changes in electrophysiological properties of neurons of the guinea-pig spinal cord

Elevated concentrations of nitric oxide (NO) and peroxynitrite (ONOO−) are present within the CNS following neurotrauma and are implicated in the pathogenesis of the accompanying neurologic deficits. We tested the hypothesis that elevated extracellular concentrations of ONOO−, introduced by the donor 3-morpholinosydnonimine (SIN-1), induce reversible axonal conduction deficits in neurons of the guinea-pig spinal cord. The compound action potential (CAP) and compound membrane potential (CMP) of excised ventral cord white matter were recorded before, during, and after, bathing the tissue (30 min) in varying concentrations (0.125–2.0 mM) of SIN-1 ( 3.75–60 μM ONOO−). The principal results were rapid onset, concentration-dependent, reductions in amplitude of the CAP (P<0.05). At a concentration of 0.25 mM of SIN-1 the reduction in CAP amplitude was fully reversible and was not accompanied by any changes in CMP. At higher concentrations of SIN-1 (≥0.5 mM) the reversibility was incomplete and there was concurrent depolarization of the CMP. These electrophysiological changes were not evident when the donor had been a priori depleted of ONOO− by uric acid or was co-administered with the ONOO− scavenger ebselen (3 mM). Immuno-fluorescence staining for nitrotyrosine (Ntyr) revealed extensive nitration of tyrosine residues in neurons exposed to higher concentrations of SIN-1. These results are the first to demonstrate that ONOO− induces reversible conduction deficits within axons of the spinal cord. The dissociation of CAP and CMP changes at low concentrations of SIN-1, when the CAP changes were reversible and there was no evidence of nitration of tyrosine residues, is consistent with ONOO−-induced alteration in Na+ channel conductance in the axolemma. The results support the view that ONOO− contributes to both reversible and non-reversible neurologic deficits following neurotrauma. The reversal of immune-mediated conduction deficits may contribute to spontaneous neurologic deficits following neurotrauma.

Activity-dependent excitability changes suggest Na+/K+ pump dysfunction in diabetic neuropathy

The present study was undertaken to evaluate the role of Na(+)/K(+) pump dysfunction in the development of diabetic neuropathy (DN). Nerve excitability techniques, which provide information about membrane potential and axonal ion channel function, were undertaken in 15 patients with established DN and in 10 patients with diabetes who had no evidence of neuropathy (DWN). Excitability parameters were recorded at baseline, and then before and after 1 min of maximal voluntary contraction (MVC) of abductor pollicis brevis. Compared to controls, CMAP amplitude was significantly decreased in DN patients with associated reductions in strength-duration time constant and refractoriness, consistent with a reduction in nodal Na(+) conductances. Following MVC for 1 min, there was an increase in normalized threshold in all diabetic patients and controls, consistent with axonal hyperpolarization. When compared to control values, the increase in threshold following MVC was significantly less in DN patients (DN group 13.1 +/- 2.2%; controls 20.4 +/- 1.9%; P < 0.05) and the rate of recovery was slower (P < 0.01). In DWN patients, CMAP amplitude was preserved, and excitability values following MVC were not significantly different to control values. The reduced threshold change and slower recovery in DN patients following MVC are likely to be secondary to Na(+)/K(+) pump dysfunction. Alteration in Na(+)/K(+) pump function, coupled with reductions in nodal Na(+) currents, may be sufficient to trigger conduction failure in DN patients and are likely to contribute to the clinical symptoms of weakness and fatigue.

Identification of FXYD protein genes in a teleost: tissue-specific expression and response to salinity change

It is increasingly clear that alterations in Na+-K+-ATPase kinetics to fit the demands in specialized cell types is vital for the enzyme to execute its different physiological roles in diverse tissues. In addition to tissue-dependent expression of isoforms of the conventional subunits, alpha and beta, auxiliary FXYD proteins appear to be essential regulatory components. The present study identified genes belonging to this family in Atlantic salmon by analysis of expressed sequence tags. Based on the conserved domain of these small membrane proteins, eight expressed FXYD isoforms were identified. Phylogenetic analysis suggests that six isoforms are homologues to the previously identified FXYD2, FXYD5, FXYD6, FXYD7, FXYD8, and FXYD9, while two additional isoforms were found (FXYD11 and FXYD12). Using quantitative PCR, tissue-dependent expression of the different isoforms was analyzed in gill, kidney, intestine, heart, muscle, brain, and liver. Two isoforms were expressed in several tissues (FXYD5 and FXYD9), while six isoforms were distributed in a discrete manner. In excitable tissues, two isoforms were highly expressed in brain (FXYD6 and FXYD7) and one in skeletal muscle (FXYD8). In osmoregulatory tissues, one isoform was expressed predominantly in gill (FXYD11), one in kidney (FXYD2), and one equally in kidney and intestine (FXYD12). Expression of several FXYD genes in kidney and gill differed between fresh water and seawater salmon, suggesting significance during osmoregulatory adaptations. In addition to identifying novel FXYD isoforms, these studies are the first to show the tissue dependence in their expression and modulation by salinity in any teleosts.

Functional Role of Extracellular Loop Cysteine Residues of the Epithelial Na+ Channel in Na+ Self-inhibition

The epithelial Na(+) channel (ENaC) is typically formed by three homologous subunits (alpha, beta, and gamma) that possess a characteristic large extracellular loop (ECL) containing 16 conserved cysteine (Cys) residues. We investigated the functional role of these Cys residues in Na(+) self-inhibition, an allosteric inhibition of ENaC activity by extracellular Na(+). All 16 Cys residues within alpha and gamma ECLs and selected beta ECL Cys residues were individually mutated to alanine or serine residues. The Na(+) self-inhibition response of wild type and mutant channels expressed in Xenopus oocytes was determined by whole cell voltage clamp. Individual mutation of eight alpha (Cys-1, -4, -5, -6, -7, -10, -13, or -16), one beta (Cys-7), and nine gamma (Cys-3, -4, -6, -7, -10, -11, -12, -13, or -16) residues significantly reduced the magnitude of Na(+) self-inhibition. Na(+) self-inhibition was eliminated by simultaneous mutations of either the last three alpha ECL Cys residues (Cys-14, -15, and -16) or Cys-7 within both alpha and gamma ECLs. By analyzing the Na(+) self-inhibition responses and the effects of a methanethiosulfonate reagent on channel currents in single and double Cys mutants, we identified five Cys pairs within the alphaECL (alphaCys-1/alphaCys-6, alphaCys-4/alphaCys-5, alphaCys-7/alphaCys-16, alphaCys-10/alphaCys-13, and alphaCys-11/alphaCys-12) and one pair within the gammaECL (gammaCys-7/gammaCys-16) that likely form intrasubunit disulfide bonds. We conclude that approximately half of the ECL Cys residues in the alpha and gamma ENaC subunits are required to establish the tertiary structure that ensures a proper Na(+) self-inhibition response, likely by formation of multiple intrasubunit disulfide bonds.

Changing salinity induces alterations in hemolymph ion concentrations and Na+ and Cl– transport kinetics of the anal papillae in the larval mosquito,Aedes aegypti

Mosquito larvae are found in diverse aquatic habitats ranging from freshwater to hypersaline water and must often deal with rapid changes in habitat salinity. We transferred larvae of Aedes aegypti from freshwater to 30% seawater, or vice versa, and measured the time course of changes in their hemolymph ion concentrations, using ion-selective microelectrodes. We also reported the Michaelis-Menten kinetics of Na(+) and Cl(-) transport by the anal papillae for the first time using the scanning ion-selective electrode technique (SIET). Hemolymph concentrations of Na(+), Cl(-) and H(+) increased within 6 h, when larvae were transferred from freshwater to seawater and decreased within 6 h, when transferred from seawater to freshwater. Kinetic parameters for Na(+) and Cl(-) transport by the anal papillae were altered after only 5 h following transfer between freshwater (FW) and 30% seawater (30%SW). The J(max) (maximum transport rate) for both ions decreased when larvae were transferred to 30%SW, whereas the K(t) (a measure of transporter affinity) increased for Na(+) transport but was unaltered for Cl(-) transport, suggesting that Na(+) and Cl(-) uptake are independent. Data reveal significant changes in ion transport by the anal papillae of mosquito larvae when they are faced with changes in external salinity such that Na(+) and Cl(-) uptake decrease in higher salinity. The alterations in Na(+) and Cl(-) uptake may be a consequence of changes in hemolymph ion levels when larvae encounter altered salinity. The rapid changes in ion transport described here compliment the previously observed long term alterations in the morphology and ultrastructure of the anal papillae.

Contribution of Renal Epithelial Na Channel (ENaC) in Clofibrate‐mediated Reduction in Blood Pressure in Female SHR

Renal epithelial sodium channels (ENaC) of the collecting duct (CD) play an important role in electrolyte transport and blood pressure (BP) regulation. ENaC activity and expression are greater in hypertension. Clofibrate (CF), a PPARα ligand, increased excretion of Na+ (UNaV) and nitrite (UNOxV) but reduced elevated BP. This study tested the hypothesis that ENaC plays no role in CF-induced alteration in Na+ transport in SHR. Female WKY and SHR rats were treated with CF (250 mg/kg) or vehicle for 7 days followed by benzamil (BM: 0.7 mg/kg), an inhibitor of ENaC. Peroxisomal β-oxidation was higher in SHR (20%) as was PPARα expression (35%) compared to WKY rats. CF increased β-oxidation and PPARα expression in WKY and SHR. CF reduced BP in SHR (11%) but not in WKY rats. Basal UNaV (4 hr) was higher in SHR (50%) and CF reduced UNaV (23%) in WKY rats but not in SHR. BM alone increased UNaV more markedly in SHR (245 vs, 18% WKY) but equally accentuated it in CF-treated WKY and SHR (2–4 fold). CF increased (2–3 fold) UNOxV in WKY and SHR but BM blunted CF-mediated increase in NO production which was more marked in SHR. ENaC protein expression was higher (33%) in SHR and CF enhanced ENaC expression in WKY but not in SHR. These data suggest that CF increased PPARα expression and reduced the elevated BP in female SHR without affecting ENaC expression or its activity consistent with a lack of PPARα expression in the CD, the primary site of action of ENaC.