The Na+/Ca2+exchanger in Alzheimer’s disease

As a pivotal player in regulating sodium (Na⁺) and calcium (Ca²⁺) homeostasis and signalling in excitable cells, the Na⁺/Ca²⁺ exchanger (NCX) is involved in many neurodegenerative disorders in which an imbalance of intracellular Ca²⁺ and/or Na⁺ concentrations occurs, including Alzheimer’s disease (AD). Although NCX has been mainly implicated in neuroprotective mechanisms counteracting Ca²⁺ dysregulation, several studies highlighted its role in the neuronal responses to intracellular Na⁺ elevation occurring in several pathophysiological conditions. Since the alteration of Na⁺ and Ca²⁺ homeostasis significantly contributes to synaptic dysfunction and neuronal loss in AD, it is of crucial importance to analyze the contribution of NCX isoforms in the homeostatic responses at neuronal and synaptic levels. Some studies found that an increase of NCX activity in brains of AD patients was correlated with neuronal survival, while other research groups found that protein levels of two NCX subtypes, NCX2 and NCX3, were modulated in parietal cortex of late stage AD brains. In particular, NCX2 positive synaptic terminals were increased in AD cohort while the number of NCX3 positive terminals were reduced. In addition, NCX1, NCX2 and NCX3 isoforms were up-regulated in those synaptic terminals accumulating amyloid-beta (Aβ), the neurotoxic peptide responsible for AD neurodegeneration. More recently, the hyperfunction of a specific NCX subtype, NCX3, has been shown to delay endoplasmic reticulum stress and apoptotic neuronal death in hippocampal neurons exposed to Aβ insult. Despite some issues about the functional role of NCX in synaptic failure and neuronal loss require further studies, these findings highlight the putative neuroprotective role of NCX in AD and open new strategies to develop new druggable targets for AD therapy.     

Structural study of the porcine NA+/H+ exchanger NHE1 gene and its 5′-flanking region

The poly(ADP-ribosyl)ation system, associated with different nuclear fractions of rat testis, has been analyzed for both pADPR and pADPR acceptor proteins. The DNase I sensitive and resistant chromatin contain 35% and 40%, respectively, of the total pADPR synthesized in intact nuclei incubated with [³²P]NAD. Moreover, the residual 25% were estimated to be associated with the nuclear matrix.Three different classes of pADPR are present in the nuclei. The longest and branched ADPribose polymers modify proteins present in the DNase I resistant (2 M NaCl extractable) chromatin and in the nuclear matrix, whereas polymers of > 20 residues interact with the components of the DNase I sensitive chromatin and oligomers of 6 ADPribose residues are bound specifically to the acid-soluble chromosomal proteins, present in isolated nuclear matrix. The main pADPR acceptor protein in all the nuclear fractions is represented by the PARP itself (auto-modification reaction). The hetero-modification reaction occurs mostly on histone H1 and core histones, that have been found associated to DNase I sensitive and resistant chromatin, respectively. Moreover, an oligo(ADP-ribosyl)ation occurs on core histones tightly-bound to the matrix associated regions (MARs) of chromatin loops.     

Human gene SLC41A1 encodes for the Na+/Mg²+ exchanger

Magnesium (Mg(2+)), the second most abundant divalent intracellular cation, is involved in the vast majority of intracellular processes, including the synthesis of nucleic acids, proteins, and energy metabolism. The concentration of intracellular free Mg(2+) ([Mg(2+)](i)) in mammalian cells is therefore tightly regulated to its optimum, mainly by an exchange of intracellular Mg(2+) for extracellular Na(+). Despite the importance of this process for cellular Mg(2+) homeostasis, the gene(s) encoding for the functional Na(+)/Mg(2+) exchanger is (are) still unknown. Here, using the fluorescent probe mag-fura 2 to measure [Mg(2+)](i) changes, we examine Mg(2+) extrusion from hSLC41A1-overexpressing human embryonic kidney (HEK)-293 cells. A three- to fourfold elevation of [Mg(2+)](i) was accompanied by a five- to ninefold increase of Mg(2+) efflux. The latter was strictly dependent on extracellular Na(+) and reduced by 91% after complete replacement of Na(+) with N-methyl-d-glucamine. Imipramine and quinidine, known unspecific Na(+)/Mg(2+) exchanger inhibitors, led to a strong 88% to 100% inhibition of hSLC41A1-related Mg(2+) extrusion. In addition, our data show regulation of the transport activity via phosphorylation by cAMP-dependent protein kinase A. As these are the typical characteristics of a Na(+)/Mg(2+) exchanger, we conclude that the human SLC41A1 gene encodes for the Na(+)/Mg(2+) exchanger, the predominant Mg(2+) efflux system. Based on this finding, the analysis of Na(+)/Mg(2+) exchanger regulation and its involvement in the pathogenesis of diseases such as Parkinson's disease and hypertension at the molecular level should now be possible.     

Intracellular pH regulation by Na⁺/H⁺ exchanger-1 (NHE1) is required for growth factor-induced mammary branching morphogenesis

Regulation of intracellular pH (pHi) and protection against cytosolic acidification is primarily a function of the ubiquitous plasma membrane Na+/H+exchanger-1 (NHE1), which uses a highly conserved process to transfer cytosolic hydrogen ions (H+) across plasma membranes in exchange for extracellular sodium ions (Na+). Growth factors, which are essential regulators of morphogenesis, have also been found to be key activators of NHE1 exchanger activity; however, the crosstalk between both has not been fully evaluated during organ development. Here we report that mammary branching morphogenesis induced by transforming growth factor-alpha (TGFα) requires PI3K-dependent NHE1-activation and subsequent pHi alkalization. Inhibiting NHE1 activity after TGFα stimulation with 10 μM of the NHE1-specific inhibitor N-Methyl-N-isobutyl Amiloride (MIA) dramatically disrupted branching morphogenesis, induced extensive proliferation, ectopic expression of the epithelial hyper-proliferative marker Keratin-6 and sustained activation of MAPK. Together these findings indicate a novel developmental signaling cascade involving TGFα>PI3K>NHE1>pHi alkalization, which leads to a permissible environment for MAPK negative feedback inhibition and thus regulated mammary branching morphogenesis.     

Export of Na+ from cells of the halotolerant microalga Dunaliella maritima: Na+/H+ antiporter or primary Na+-pump?

Transport of Na+ and K+ ions through the plasma membrane of intact cells of the halotolerant microalga Dunaliella maritima Massjuk was studied. Ion fluxes through the plasma membrane were induced by hyperosmotic shock (uptake of Na+ by the cells is transformed into extrusion of Na+) or by addition of K+ to a suspension of K+-deficient cells (uptake of K+ by the cells is associated with extrusion of Na+). The pathway of Na+ extrusion from the D. maritima cells does not depend on the direction or value of the proton gradient on the plasma membrane. In particular, the efficiency of Na+ extrusion was not changed at extracellular pH values varying from 6.0 to 8.0. The protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) (20 microM) and the H+-ATPase inhibitor N,N-dicyclohexyl carbodiimide (DCCD) (25 and 100 microM) inhibited accumulation of K+ by the cells but did not influence Na+ extrusion. Significant acidification of the medium did not induce a net current of Na+ from the cells through a Na+/H+ antiporter. The data indicate that the Na+/H+ antiporter of the plasma membrane of D. maritima is not responsible for Na+ extrusion from the cells. These results can be explained by the involvement of a primary electrogenic Na+ pump (a Na+-transporting ATPase) in Na+ transfer through the plasma membrane of this alga.     

Ontogeny of the Na+−H+ exchanger in rat ileal brush-border membrane vesicles

Summary The developmental maturation of Na⁺–H⁺ antiporter was determined using a well-validated brush-border membrane vesicles (BBMV's) technique. Na⁺ uptake represented transport into an osmotically sensitive intravesicular space as evidenced by an osmolality study at equilibrium. An outwardly directed pH gradient (pH inside/pH outside=5.2/7.5) significantly stimulated Na⁺ uptake compared with no pH gradient conditions at all age groups; however, the magnitude of stimulation was significantly different between the age groups. Moreover, the imposition of greater pH gradient across the vesicles resulted in marked stimulation of Na⁺ uptake which increased with advancing age. Na⁺ uptake represented an electroneutral process.The amiloride sensitivity of the pH-stimulated Na⁺ uptake was investigated using [amiloride] 10^–2–10^–5 m. At 10^–3 m amiloride concentration, Na⁺ uptake under pH gradient conditions was inhibited 80, 45, and 20% in BBMV's of adolescent, weanling and suckling rats, respectively. Kinetic studies revealed aK _m for amiloride-sensitive Na⁺ uptake of 21.8±6.4, 24.9±10.9 and 11.8±4.17mm andV _max of 8.76±1.21, 5.38±1.16 and 1.99±0.28 nmol/mg protein/5 sec in adolescent, weanling and suckling rats, respectively. The rate of pH dissipation, as determined by the fluorescence quenching of acridine orange, was similar across membrane preparation of all age groups studied. These findings suggest for the first time the presence of an ileal brush-border membrane Na⁺–H⁺ antiporter system in all ages studied. This system exhibits changes in regard to amiloride sensitivity and kinetic parameters.     

The molecular chaperone Hsp90α is required for meiotic progression of spermatocytes beyond pachytene in the mouse

The molecular chaperone Hsp90 has been found to be essential for viability in all tested eukaryotes, from the budding yeast to Drosophila. In mammals, two genes encode the two highly similar and functionally largely redundant isoforms Hsp90α and Hsp90β. Although they are co-expressed in most if not all cells, their relative levels vary between tissues and during development. Since mouse embryos lacking Hsp90β die at implantation, and despite the fact that Hsp90 inhibitors being tested as anti-cancer agents are relatively well tolerated, the organismic functions of Hsp90 in mammals remain largely unknown. We have generated mouse lines carrying gene trap insertions in the Hsp90α gene to investigate the global functions of this isoform. Surprisingly, mice without Hsp90α are apparently normal, with one major exception. Mutant male mice, whose Hsp90β levels are unchanged, are sterile because of a complete failure to produce sperm. While the development of the male reproductive system appears to be normal, spermatogenesis arrests specifically at the pachytene stage of meiosis I. Over time, the number of spermatocytes and the levels of the meiotic regulators and Hsp90 interactors Hsp70-2, NASP and Cdc2 are reduced. We speculate that Hsp90α may be required to maintain and to activate these regulators and/or to disassemble the synaptonemal complex that holds homologous chromosomes together. The link between fertility and Hsp90 is further supported by our finding that an Hsp90 inhibitor that can cross the blood-testis barrier can partially phenocopy the genetic defects.     

The pipe and the pinwheel: Is pressure an effective stimulus for the 9 + 0 primary cilium?

Almost universally, the effective stimulus for mammalian 9+0 primary cilia has been taken to be bending. In this article I point out that in several physiological contexts there is great advantage in detecting pressure differences across the cell wall, i.e. axially directed forces pushing fluid to and fro through the hollow cilium and its basal body beneath. The form of the cilium--a fluid-filled pipe that connects to an intricate pinwheel-shaped basal body--is well configured for detecting fluid flow. Pressure-detection calls for compressible elements within the cell, but it effectively matches form and function in a range of cases. The "pipe and pinwheel" scheme suggests that the bulbous swellings commonly found near the tip of some primary cilia are compliant, pressure-sensitive elements which act like the bulb of an eye-dropper. In looking exclusively at the bending of cilia, we might be missing aspects of a dual-stimulus system.     

What is the primary function of the early teleost gill? Evidence for Na+/NH+4 exchange in developing rainbow trout (Oncorhynchus mykiss)

Post-hatch fishes lack a functional gill and use cutaneous surfaces for exchange with the surrounding environment. The ionoregulatory hypothesis posits that ionoregulation is the first physiological process to be limited by cutaneous exchange, necessitating its shift to the gills. We hypothesized that the ontogeny of branchial ammonia excretion (J_amm) is coupled to Na⁺ uptake () in accordance with the current model for exchange in freshwater. Using divided chambers, branchial and cutaneous J_amm, and oxygen consumption (MO₂) by larval rainbow trout were assessed. Following hatch, the skin accounted for 97% and 86% of total J_amm and , respectively. J_amm and shifted to the gills simultaneously at 15 days post-hatch (dph) and were highly correlated (R² = 0.951) at the gills, but not the skin, over development. Contrastingly, MO₂ shifted significantly later at 27 dph, in agreement with the ionoregulatory hypothesis. Moreover, the mRNA expression and/or enzymatic activity of Rhesus proteins, Na⁺/H⁺-exchanger, H⁺-ATPase, Na⁺/K⁺-ATPase and carbonic anhydrase, all key components of the -exchange system, increased in the gills over larval development. We propose that the ontogeny of branchial occurs as exchange and provide evidence for a novel element to the ionoregulatory hypothesis, the excretion of potentially lethal metabolic ammonia.     

SKIP is required for TGF-β1-induced epithelial mesenchymal transition and migration in transformed keratinocytes

Transforming growth factor-β1 (TGF-β1) potently induces the epithelial-mesenchymal transition (EMT) during tumoral progression. Although Sky-interacting protein (SKIP) regulates TGF-β1-induced Smad activation, its role in the induction of cell malignance remains uncertain. We found that TGF-β1 increases SKIP expression in PDV cells. In cells stably transfected with SKIP antisense, AS-S, Smad3 activation decreased, along with an inhibition of TGF-β1-induced EMT, and the cells were sensitized to the TGF-β1-dependent inhibition of proliferation. Also, AS-S cells showed a weaker migration and invasion response. Moreover, TGF-β1-induced urokinase-type plasminogen activator expression was inhibited, concomitantly with a TGF-β1-independent increment of the plasminogen-activator inhibitor-1 expression. Thus, these results suggest that SKIP is required for EMT and invasiveness induced by TGF-β1 in transformed cells.     

Influence of initial respiratory status on the short-and long-term activity of the trout red blood cell β-adrenergic Na+/H+ exchanger

The effects of ambient O₂ partial pressure and CO₂ partial pressure on the intensity of rainbow trout (Oncorhynchus mykiss) red blood cell -adrenergic Na⁺/H⁺ exchange were investigated. This was accomplished in vitro by continuously monitoring whole blood extracellular pH, partial pressures of O₂ and CO₂ and by measuring red blood cell water content and Na⁺ concentration before and 30 min after the addition of a catecholamine mixture (final nominal concentrations: 250 nmol·l^-1 adrenaline and 20 nmol·l^-1 noradrenaline). The experiments were performed under six different initial conditions combining two ambient partial pressures of CO₂ (1.50 and 6.75 torr) and three ambient partial pressures of O₂ (15, 30 and 150 torr). The activation of red blood cell Na⁺/H⁺ exchange (as indicated by marked reductions of whole blood pH) was followed by transient reductions in blood partial pressures of CO₂ and O₂ (2 min) resulting from the shift of the CO₂/HCO₃ ^- equilibrium within the cell and the subsequent binding of O₂ to the haemoglobin. The initial reduction in blood CO₂ partial pressure was followed by a rise reflecting the titration of plasma HCO₃ ^- by extruded H⁺. At low partial pressure of CO₂ (1.50 torr) there was a pronounced stimulatory effect of hypoxia on the initial intensity of the extracellular acidification (5 min), whereas at high CO₂ partial pressure (6.75 torr) hypoxia actually lowered the extent of the initial acidification. In all cases, Na⁺/H⁺ exchange activation was accompanied by increases in cell water content and red blood cell Na⁺ levles when measured 30 min after addition of catecholamines. Both hypercapnia and hypoxia increased the magnitude of these changes although the largest changes in cell water content and Na⁺ levels were observed under hypercapnic conditions. Thus, the long-term activity (as determined by measuring cell water and Na⁺ levels) of the Na⁺/H⁺ exchanger was enhanced both by hypercapnia and hypoxia regardless of the initial CO₂ partial pressure. The initial activity (5 min), on the other hand, although stimulated by hypercapnia was influenced by hypoxia in opposing directions depending upon the initial CO₂ partial pressure of the blood.Abbreviations RBC red blood cell(s) - Hb haemoglobin - pHe extracellular pH - P _bCO₂ blood partial pressure of CO₂ - P _bO₂ blood partial pressure of O₂     

Identification of the renal Na+/H+ exchanger with N,N′-dicyclohexylcarbodiimide (DCCD) and amiloride analogues

Summary Dicyclohexylcarbodiimide (DCCD) and the 5-ethylisopropyl-6-bromo-derivative of amiloride (Br-EIPA) have been used as affinity and photoaffinity labels of the Na⁺/H⁺ exchanger in rat renal brush-border membranes. Intravesicular acidification by the Na^–/H⁺ exchanger was irreversibly inhibited after incubation of vesicles for 30 min with DCCD. The substrate of the antiporter, Na⁺, and the competitive inhibitor, amiloride, protected from irreversible inhibition. The Na⁺-dependent transport systems for sulfate, dicarboxylates, and neutral, acidic, and basic amino acids were inhibited by DCCD, but not protected by amiloride. An irreversible inhibition of Na⁺/H⁺ exchange was also observed when brush-border membrane vesicles were irradiated in the presence of Br-EIPA. Na⁺ and Li⁺ protected. [¹⁴C]-DCCD was mostly incorporated into three brush-border membrane polypeptides with apparent molecular weights of 88,000, 65,000 and 51,000. Na⁺ did not protect but rather enhanced labeling. In contrast, amiloride effectively decreased the labeling of the 65,000 molecular weight polypeptide. In basolateral membrane vesicles one band was highly labeled by [¹⁴C]-DCCD that was identified as the -subunit of the Na⁺, K⁺-ATPase. [¹⁴C]-Br-EIPA was mainly incorporated into a brushborder membrane polypeptide with apparent molecular weight of 65,000. Na⁺ decreased the labeling of this protein. Similar to the Na⁺/H⁺ exchanger this Na⁺-protectable band was absent in basolateral membrane vesicles. We conclude that a membrane protein with an apparent molecular weight of 65,000 is involved in rat renal Na⁺/H⁺ exchange.