Endogenous NA Research Articles

The NH 2-terminus of K +-Cl − cotransporter 3a is essential for up-regulation of Na +,K +-ATPase activity

K +-Cl − cotransporter-3 has two major amino terminal variants, KCC3a and KCC3b. In LLC-PK1 cells, exogenously expressed KCC3a co-immunoprecipitated with endogenous Na +,K +-ATPase α1-subunit (α1NaK), accompanying significant increases of the Na +,K +-ATPase activity. Exogenously expressed KCC3b did not co-immunoprecipitate with endogenous α1NaK inducing no change of the Na +,K +-ATPase activity. A KCC inhibitor attenuated the Na +,K +-ATPase activity in rat gastric mucosa in which KCC3a is predominantly expressed, while it had no effects on the Na +,K +-ATPase activity in rat kidney in which KCC3b is predominantly expressed. In these tissue samples, KCC3a co-immunoprecipitated with α1NaK, while KCC3b did not. Our results suggest that the NH 2-terminus of KCC3a is a key region for association with α1NaK, and that KCC3a but not KCC3b can regulate the Na +,K +-ATPase activity.

P.1.d.001 Marinobufagenin, an endogenous Na pump inhibitor, is implicated in social behavior of mice

P.1.d.001 Marinobufagenin, an endogenous Na pump inhibitor, is implicated in social behavior of mice

Isoform specificity of cardiac glycosides binding to human Na +,K +-ATPase α 1β 1, α 2β 1 and α 3β 1

Cardiac glycosides inhibit the Na +,K +-ATPase and are used for the treatment of symptomatic heart failure and atrial fibrillation. In human heart three isoforms of Na +,K +-ATPase are expressed: α 1β 1, α 2β 1 and α 3β 1. It is unknown, if clinically used cardiac glycosides differ in isoform specific affinities, and if the isoforms have specific subcellular localization in human cardiac myocytes. Human Na +,K +-ATPase isoforms α 1β 1, α 2β 1 and α 3β 1 were expressed in yeast which has no endogenous Na +,K +-ATPase. Isoform specific affinities of digoxin, digitoxin, β-acetyldigoxin, methyldigoxin and ouabain were assessed in [³H]-ouabain binding assays in the absence or presence of K + (each n = 5). The subcellular localizations of the Na +,K +-ATPase isoforms were investigated in isolated human atrial cardiomyocytes by immunohistochemistry. In the absence of K +, methyldigoxin (α 1 > α 3 > α 2) and ouabain (α 1 = α 3 > α 2) showed distinct isoform specific affinities, while for digoxin, digitoxin and β-acetyldigoxin no differences were found. In the presence of K +, also digoxin (α 2 = α 3 > α 1) and β-acetyldigoxin (α 1 > α 3) had isoform specificities. A comparison between the cardiac glycosides demonstrated highly different affinity profiles for the isoforms. Immunohistochemistry showed that all three isoforms are located in the plasma membrane and in intracellular membranes, but only α 1β 1 and α 2β 1 are located in the T-tubuli. Cardiac glycosides show distinct isoform specific affinities and different affinity profiles to Na +,K +-ATPase isoforms which have different subcellular localizations in human cardiomyocytes. Thus, in contrast to current notion, different cardiac glycoside agents may significantly differ in their pharmacological profile which could be of hitherto unknown clinical relevance.

Marinobufagenin enhances cardiac contractility in mice with ouabain-sensitive α1Na+-K+-ATPase

Endogenous Na(+) pump inhibitors are thought to play important (patho)physiological roles and occur in two different chemical forms in the mammalian circulation: cardenolides, such as ouabain, and bufadienolides, such as marinobufagenin (MBG). Although all alpha Na(+)-K(+)-ATPase isoforms (alpha(1-4)) are sensitive to ouabain in most species, in rats and mice the ubiquitously expressed alpha(1) Na(+)-K(+)-ATPase is resistant to ouabain. We have previously shown that selective modification of the putative ouabain binding site of either the alpha(1) or alpha(2) Na(+)-K(+)-ATPase subunit in mice substantially alters the cardiotonic influence of exogenously applied cardenolides. To determine whether the ouabain binding site also interacts with MBG and if this interaction plays a functional role, we evaluated cardiovascular function in alpha(1)-resistant/alpha(2)-resistant (alpha(1)(R/R)alpha(2)(R/R)), alpha(1)-sensitive/alpha(2)-resistant (alpha(1)(S/S)alpha(2)(R/R)), and alpha(1)-resistant/alpha(2)-sensitive mice (alpha(1)(R/R)alpha(2)(S/S), wild type). Cardiovascular indexes were evaluated in vivo by cardiac catheterization at baseline and during graded infusions of MBG. There were no differences in baseline measurements of targeted mice, indicating normal hemodynamics and cardiac function. MBG at 0.025, 0.05, and 0.1 nmol*min(-1)*g body wt(-1) significantly increased cardiac performance to a greater extent in alpha(1)(S/S)alpha(2)(R/R) compared with alpha(1)(R/R)alpha(2)(R/R) and wild-type mice. The increase in LVdP/dt(max) in alpha(1)(S/S)alpha(2)(R/R) mice was greater at higher concentrations of MBG compared with both alpha(1)(R/R)alpha(2)(R/R) and alpha(1)(R/R)alpha(2)(S/S) mice (P < 0.05). These results suggest that MBG interacts with the ouabain binding site of the alpha(1) Na(+)-K(+)-ATPase subunit and can thereby influence cardiac inotropy.

Cloning and Sequence Analysis of the Voltage-Gated Muscle Na+ Channel from the Poison Dart Frog Phyllobates aurotaenia

Poison dart frogs of the genus Phyllobates secrete lipophilic alkaloid toxins through their skin that were used by Colombian Amerindians to poison the tips of blowdarts. One of the most potent toxins identified is batrachotoxin (BTX) which is an activator of voltage-gated Na+ channels. BTX causes sustained opening of these channels by shifting the voltage-dependent activation to more hyperpolarized potentials and by disabling both fast and slow inactivation. It also alters pore conductance and selectivity. Endogenous Na+ channels of the poison arrow frog have been proposed to be insensitive to lethal amounts of BTX. In this project we aim to identify what confers BTX insensitivity to Na+ channels of the host frog Phyllobates aurotaenia, therefore we cloned its skeletal muscle NaV channel. Total RNA from skeletal muscle of Phyllobates aurotaenia was isolated and cDNA was obtained with degenerate primers.The 1819 amino acids sequence shares 72% sequence identity with the rat Na+ channel NaV1.4, and 73% with that of the snake Thamnophis sirtalis. The TMs are extremely well conserved (87%) with absolute conservation of S4 in all domains. The N-and C-termini as well as the cytoplasmic linkers between domains are more divergent. The D3-D4 linker containing the IFM motif is highly conserved except for Q1348E and K1350P. The DEKA-motif is also absolutely conserved as are the GGGS gating hinge and the QGFS motifs. BTX is thought to bind in the pore region, from the selectivity filter ring to the pore lining S6 TMs. We have identified two S to A mutations flanking the gating-hinge in domains 1 and 3 that may participate in toxin-insensitivity of the Phyllobates channel by impairing the binding of BTX. Supported by NIH GM68044(AMC) and GM30376(FB) and by COLCIENCIAS1106-12-13836(LF).

Identification and Function of a Cytoplasmic K+ Site of the Na+, K+-ATPase

A cytoplasmic nontransport K(+)/Rb(+) site in the P-domain of the Na(+), K(+)-ATPase has been identified by anomalous difference Fourier map analysis of crystals of the [Rb(2)].E(2).MgF(4)(2-) form of the enzyme. The functional roles of this third K(+)/Rb(+) binding site were studied by site-directed mutagenesis, replacing the side chain of Asp(742) donating oxygen ligand(s) to the site with alanine, glutamate, and lysine. Unlike the wild-type Na(+), K(+)-ATPase, the mutants display a biphasic K(+) concentration dependence of E(2)P dephosphorylation, indicating that the cytoplasmic K(+) site is involved in activation of dephosphorylation. The affinity of the site is lowered significantly (30-200-fold) by the mutations, the lysine mutation being most disruptive. Moreover, the mutations accelerate the E(2) to E(1) conformational transition, again with the lysine substitution resulting in the largest effect. Hence, occupation of the cytoplasmic K(+)/Rb(+) site not only enhances E(2)P dephosphorylation but also stabilizes the E(2) dephosphoenzyme. These characteristics of the previously unrecognized nontransport site make it possible to account for the hitherto poorly understood trans-effects of cytoplasmic K(+) by the consecutive transport model, without implicating a simultaneous exposure of the transport sites toward the cytoplasmic and extracellular sides of the membrane. The cytoplasmic K(+)/Rb(+) site appears to be conserved among Na(+), K(+)-ATPases and P-type ATPases in general, and its mode of operation may be associated with stabilizing the loop structure at the C-terminal end of the P6 helix of the P-domain, thereby affecting the function of highly conserved catalytic residues and promoting helix-helix interactions between the P- and A-domains in the E(2) state.

Marinobufagenin enhances cardiac contractility in mice with ouabain‐sensitive α1 Na,K‐ATPase (NKA)

Endogenous Na+ pump inhibitors are thought to play important (patho)physiological roles, and occur in two different chemical forms in the mammalian circulation: cardenolides, such as ouabain, and bufadienolides, such as marinobufagenin (MBG). While all αNKA isoforms are sensitive to ouabain in most species, in rats and mice the ubiquitously expressed α1NKA is resistant to ouabain. We have previously shown that selective modification of the putative ouabain‐binding site of either the α1 or α2 NKA subunit in mice substantially alters the cardiotonic influence of exogenously applied cardenolides. To determine whether the ouabain‐binding site also interacts with MBG and if this interaction plays a functional role, we evaluated cardiovascular function in α1‐resistant/α2‐resistant (α1R/Rα2R/R), α1‐sensitive/α2‐resistant (α1S/Sα2R/R), and α1‐resistant/α2‐sensitive mice (α1R/Rα2S/S, wild type). Cardiovascular indices were evaluated in vivo by cardiac catheterization at baseline and during graded infusions of MBG. There were no differences in baseline measurements of targeted mice, indicating normal hemodynamics and cardiac function. As shown in the graph, MBG increased cardiac performance to a greater extent in α1S/Sα2R/R compared to α1R/Rα2R/R and wild type mice. These data indicate that MBG interacts with the ouabain‐binding site of the α1NKA subunit and can thereby influence cardiac inotropy.

The Expression of NMDA Receptor Subunits in Cerebral Cortex and Hippocampus is Differentially Increased by Administration of Endobain E, a Na+, K+-ATPase Inhibitor

Previous studies showed that endobain E, an endogenous Na+, K+-ATPase inhibitor, decreases dizocilpine binding to NMDA receptor in isolated membranes. The effect of endobain E on expression of NMDA receptor subunits in membranes of rat cerebral cortex and hippocampus was analyzed by Western blot. Two days after administration of 10 mul endobain E (1 microl = 29 mg fresh tissue) NR1 subunit expression enhanced 5-fold and 2.5-fold in cerebral cortex and hippocampus, respectively. NR2A subunit expression increased 2-fold in cerebral cortex and 1.5-fold in hippocampus. The level of NR2B subunit raised 3-fold in cerebral cortex but remained unaltered in hippocampus. NR2C subunit expression was unaffected in either area. NR2D subunit enhanced 1.6 and 2.1-fold for cerebral cortex and hippocampus, respectively. Results indicate that endogenous Na+, K+-ATPase inhibitor endobain E differentially modifies the expression of NMDA receptor subunits.

The Small Molecule NS11021 Is a Potent and Specific Activator of Ca2+-Activated Big-Conductance K+ Channels

Large-conductance Ca(2+)- and voltage-activated K(+) channels (Kca1.1/BK/MaxiK) are widely expressed ion channels. They provide a Ca(2+)-dependent feedback mechanism for the regulation of various body functions such as blood flow, neurotransmitter release, uresis, and immunity. In addition, a mitochondrial K(+) channel with KCa1.1-resembling properties has been found in the heart, where it may be involved in regulation of energy consumption. In the present study, the effect of a novel NeuroSearch compound, 1-(3,5-bis-trifluoromethyl-phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea (NS11021), was investigated on cloned KCa1.1 expressed in Xenopus laevis oocytes and mammalian cells using electrophysiological methods. NS11021 at concentrations above 0.3 microM activated KCa1.1 in a concentration-dependent manner by parallel-shifting the channel activation curves to more negative potentials. Single-channel analysis revealed that NS11021 increased the open probability of the channel by altering gating kinetics without affecting the single-channel conductance. NS11021 (10 microM) influenced neither a number of cloned Kv channels nor endogenous Na(+) and Ca(2+) channels (L- and T-type) in guinea pig cardiac myocytes. In conclusion, NS11021 is a novel KCa1.1 channel activator with better specificity and a 10 times higher potency compared with the most broadly applied KCa1.1 opener, NS1619. Thus, NS11021 might be a valuable tool compound when addressing the physiological and pathophysiological roles of KCa1.1 channels.

Expression and characterization of the Na+/H+ exchanger in the mammalian myocardium

We examined two expression systems for studying the Na(+)/H(+) exchanger in the mammalian myocardium. Mammalian NHE1 with a hemagglutinin (HA) tag and was cloned behind the alpha myosin heavy chain promoter. Transgenic mice were made with wild type NHE1 protein or with a hyperactive NHE1 protein mutated at the calmodulin-binding domain. Three lines of transgenic mice were made of each cDNA with expression levels of each type varying from high to low. Higher levels and activity of the Na(+)/H(+) exchanger were associated with decreased long-term survival of mice, and with dilated or hypertrophic cardiomyopathy. The exogenous NHE1 protein was present in freshly made cardiomyocytes from transgenic mice, however, expression from the alpha myosin heavy chain promoter declined rapidly and little exogenous NHE1 was apparent on the fourth day after cardiomyocyte isolation. To express NHE1 protein in isolated cardiomyocytes, we transferred a mutated form of the protein into an adenoviral expression system. Infection of neonatal rat cardiomyocytes resulted in robust expression of the exogenous NHE1 protein. The mutant form of the NHE1 protein could be distinguished from the endogenous Na(+)/H(+) exchanger by its resistance to inhibition by amiloride analogs. Our results suggest that for in vivo studies on intact hearts and animals, expression in transgenic mice is an appropriate system, however for long-term studies on cardiomyocytes, this model is inappropriate due to waning expression from the alpha myosin heavy chain promoter. Therefore, infection by adenovirus is a superior system for long-term studies on cardiomyocytes in culture.

NaCl effects growth, ions and water status of Tomato (Lycopersicon esculentum) seedlings

Tomato seedlings (Lycopersicon esculentum Mill, cv Chibli F1) were subjected to different NaCl treatments (0, 25, 50 and 100 mM) during 10 days. Under low salinity (25 mM NaCl), tomato preserved a normal growth activity, associated with maintain of the tissues water status and a high selectivity for K+ over Na+. Under high salinity (50 and 100 mM), endogenous Na+ and CI− accumulations were contradicted by a decrease of K+ and NO3 − contents. Our results support the existence of a competition between K+/Na+ and NO3 −CI− at the level of uptake and storage process. The high salt stress (50 and 100 mM) emphasized a typical glycophytic behavior; displaying i) a severe decline in plant growth activity, ii) the low Na+ amounts compared to CI− in the leaves, make an evidence for Na+ extrusion from the leaves to the stems-petioles and roots, and iii) an inaptitude to use Na+ instead of K+ to carry out osmotic adjustments at high salinity (100 mM NaCl).

The Epithelial Na+ Channel Is Inhibited by a Peptide Derived from Proteolytic Processing of Its α Subunit

Epithelial sodium channels (ENaCs) mediate Na(+) entry across the apical membrane of high resistance epithelia that line the distal nephron, airway and alveoli, and distal colon. These channels are composed of three homologous subunits, termed alpha, beta, and gamma, which have intracellular amino and carboxyl termini and two membrane-spanning domains connected by large extracellular loops. Maturation of ENaC subunits involves furin-dependent cleavage of the extracellular loops at two sites within the alpha subunit and at a single site within the gamma subunit. The alpha subunits must be cleaved twice, immediately following Arg-205 and Arg-231, in order for channels to be fully active. Channels lacking alpha subunit cleavage are inactive with a very low open probability. In contrast, channels lacking both alpha subunit cleavage and the tract alphaAsp-206-Arg-231 are active when expressed in oocytes, suggesting that alphaAsp-206-Arg-231 functions as an inhibitor that stabilizes the channel in the closed conformation. A synthetic 26-mer peptide (alpha-26), corresponding to alphaAsp-206-Arg-231, reversibly inhibits wild-type mouse ENaCs expressed in Xenopus oocytes, as well as endogenous Na(+) channels expressed in either a mouse collecting duct cell line or primary cultures of human airway epithelial cells. The IC(50) for amiloride block of ENaC was not affected by the presence of alpha-26, indicating that alpha-26 does not bind to or interact with the amiloride binding site. Substitution of Arg residues within alpha-26 with Glu, or substitution of Pro residues with Ala, significantly reduced the efficacy of alpha-26. The peptide inhibits ENaC by reducing channel open probability. Our results suggest that proteolysis of the alpha subunit activates ENaC by disassociating an inhibitory domain (alphaAsp-206-Arg-231) from its effector site within the channel complex.

Modulation of Aspartate Release by Ascorbic Acid and Endobain E, an Endogenous Na+, K+-ATPase Inhibitor

The isolation of a soluble brain fraction which behaves as an endogenous ouabain-like substance, termed endobain E, has been described. Endobain E contains two Na+, K+ -ATPase inhibitors, one of them identical to ascorbic acid. Neurotransmitter release in the presence of endobain E and ascorbic acid was studied in non-depolarizing (0 mM KCl) and depolarizing (40 mM KCl) conditions. Synaptosomes were isolated from cerebral cortex of male Wistar rats by differential centrifugation and Percoll gradient. Synaptosomes were preincubated in HEPES-saline buffer with 1 mM D-[3H]aspartate (15 min at 37 degrees C), centrifuged, washed, incubated in the presence of additions (60 s at 37 degrees C) and spun down; radioactivity in the supernatants was quantified. In the presence of 0.5-5.0 mM ascorbic acid, D-[3H]aspartate release was roughly 135-215% or 110-150%, with or without 40 mM KCI, respectively. The endogenous Na+, K+ -ATPase inhibitor endobain E dose-dependently increased neurotransmitter release, with values even higher in the presence of KCl, reaching 11-times control values. In the absence of KCl, addition of 0.5-10.0 mM commercial ouabain enhanced roughly 100% D-[3H]aspartate release; with 40 mM KCl a trend to increase was recorded with the lowest ouabain concentrations to achieve statistically significant difference vs. KCl above 4 mM ouabain. Experiments were performed in the presence of glutamate receptor antagonists. It was observed that MPEP (selective for mGluR5 subtype), failed to decrease endobain E response but reduced 50-60% ouabain effect; LY-367385 (selective for mGluR1 subtype) and dizocilpine (for ionotropic NMDA glutamate receptor) did not reduce endobain E or ouabain effects. These findings lead to suggest that endobain E effect on release is independent of metabotropic or ionotropic glutamate receptors, whereas that of ouabain involves mGluR5 but not mGluR1 receptor subtype. Assays performed at different temperatures indicated that in endobain E effect both exocytosis and transporter reversion are involved. It is concluded that endobain E and ascorbic acid, one of its components, due to their ability to inhibit Na+, K+ -ATPase, may well modulate neurotransmitter release at synapses.

Antioxidant Enzyme Responses to NaCl Stress in Cassia angustifolia

Seeds of Cassia angustifolia Vahl. were subjected to 0, 20, 50, 100 mM NaCl for 7 d in order to study the effect of salt stress on growth parameters, endogenous Na+ and Cl− concentrations, antioxidant system, lipid peroxidation, hydrogen peroxide, and proline contents. Salinity affected all of the considered parameters and caused a great reduction in plant biomass. The root and shoot length, fresh and dry mass and germination percentage were inhibited by NaCl treatments. These changes were associated with an increase in the Na+ and Cl− contents in the seedlings and increased activities of superoxide dismutase, catalase, peroxidase, and polyphenol oxidase. The increased enzyme activity coincided with decreased ascorbate content and enhanced H2O2 and proline content.

Temporal changes in intestinal Na +, K +-ATPase activity and in vitro responsiveness to cortisol in juvenile chinook salmon

Seasonal changes in endogenous Na +, K +-ATPase activity were measured in pyloric ceca and posterior intestine of juvenile chinook salmon ( Oncorhynchus tshawytscha) maintained in fresh water over 18 months. In tissues from these same fish, the in vitro responsiveness of Na +, K +-ATPase activity to 10 μg cortisol/ml was assessed. There were pronounced increases in endogenous Na +, K +-ATPase activity in summer for both intestinal regions, in underyearlings and yearlings. In pyloric ceca, a significant positive response of Na +, K +-ATPase activity to cortisol, in vitro, was restricted to the months preceding increases in endogenous Na +, K +-ATPase and the month afterward. Na +, K +-ATPase activity of the posterior intestine was only responsive to cortisol in underyearlings in the period before the peak in endogenous enzyme activity. At a time when explants were responsive to cortisol, in vitro exposure to 0.1–10 μg cortisol/ml resulted in dose-dependent elevations of Na +, K +-ATPase activity over controls (0 μg cortisol/ml). The results show that the intestine exhibits increased enzymatic potential for water absorption that is indicative of parr–smolt transformation. Alterations in tissue responsiveness to cortisol may contribute to these changes in Na +, K +-ATPase activity of pyloric ceca.

ELEVATION OF [NA+]I PROTECTS ENDOTHELIAL CELLS FROM APOPTOSIS TRIGGERED BY [3H]-DECAY-INDUCED DNA DAMAGE

Objective: Endothelial cell damage contributes to vascular remodeling seen in hypertension and other cardiovascular diseases. Previously, we reported that elevation of the [Na+]i/[K+]i ratio protects vascular smooth muscle cells against apoptosis. This study examines the role of intracellular monovalent cations in apoptosis of porcine aorta endothelial cells (PAEC). Design and Methods: Apoptosis in cultured freshly-isolated PAEC was estimated by MTT assay and caspase-3 activity. [14C]-urea available space and atomic absorption spectrophotometry were employed to measure cell volume and intracellular Na and K content, respectively. Results: 24-hr treatment of PAEC with 5 uCi/ml of [3H]-thymidine led to 2-fold attenuation of MTT staining and 10-fold elevation of caspase-3 activity. Apoptosis in [3H]-thymidine-treated cells was abolished by nonradioactive thymidine, the pan-caspase inhibitor z-VAD.fmk and by Na,KATPase inhibition in K+ free medium. Incubation of PAEC in K+ free medium led to 10-fold elevation of Na + i and to 8-fold decrease of K + i. Inhibition of the Na,K-ATPase in K + free Na + depleted medium resulted in the same loss of K + i, but led to modest (2-fold) elevation of Na + i and did not protect PAEC against apoptosis. Conclusions: Extensive DNA labeling with [3H]-thymidine is sufficient to induce apoptosis in PAEC. Elevation of [Na+]i protects PAEC from apoptosis triggered by [3H]-decay-induced DNA damage at a step upstream of caspase-3. These data should be considered under analysis of the involvement of endogenous Na+,K+ ATPase inhibitors and other potent Na + i raising compounds in endothelial cell survival and vascular remodeling seen in hypertension.

Allosteric modulation of [ 3H]dizocilpine binding to N-methyl- d-aspartate receptor by an endogenous Na +, K +-ATPase inhibitor: dependence on receptor activation

An endogenous Na +, K +-ATPase inhibitor, termed endobain E, has been isolated from rat brain and proved to decrease [ 3H]dizocilpine binding to cerebral cortex N-methyl- d-aspartate (NMDA) receptor, an effect independent of sodium pump activity. The purpose of this study was to disclose the mechanism of [ 3H]dizocilpine binding reduction by endobain E by performing saturation, kinetic and competitive assays. In saturation binding assays, endobain E increased K d without modifying B max value. To determine whether competitive or allosteric interaction was involved, kinetics of [ 3H]dizocilpine binding to cerebral cortex membranes was studied. Endobain E increased [ 3H]dizocilpine dissociation rate constant and induced an initial fast phase, without modifying association rate constant, indicating an allosteric interaction. In competitive [ 3H]dizocilpine binding assays, no additive effect was observed with endobain E plus competitive antagonists for glutamate or glycine sites (2-amino-5-phosphonopentanoic acid (AP-5) and 7-chlorokynurenic acid, respectively), indicating that coagonist site blockade interferes with endobain E effect. However, the higher glutamate and glycine concentration, the greater its effect. Endobain E binding reduction was partially additive with that induced by ketamine or Mg 2+ (receptor-associated channel blockers). Results suggest that the greater the channel activation by glutamate and glycine, the greater endobain E allosteric effect. Furthermore, as ketamine and Mg 2+ interfere with endobain E effect, this factor most likely binds to the inner surface of the NMDA associated channel.

Hypothalamic digoxin – central role in conscious perception, neuroimmunoendocrine integration and coordination of cellular function – relation to hemispheric dominance

A family with a high prevalence of Parkinson’s disease, schizophrenia, neoplasms, syndrome-X, rheumatoid arthritis and epilepsy has been described. The psychological behavioural patterns of the family were as follows – creativity and high IQ, hypersexual behaviour, reduced appetite and eating behaviour, insomnia and reduced sleep patterns, increased tendency for spirituality, increased tendency for addiction, less of bonding and affectionate behaviour and left handedness. Digoxin, an endogenous Na +–K + ATPase inhibitor secreted by the hypothalamus, was found to be elevated and RBC membrane Na +–K + ATPase activity was found to be reduced in all the disorders and in the indexed family studied. Hypothalamic digoxin can modulate conscious perception and its dysfunction may lead to schizophrenia. Digoxin can also preferentially upregulate tryptophan transport over tyrosine resulting in increased levels of depolarising tryptophan catabolites – serotonin, quinolinic acid, strychnine and nicotine and decreased levels of hyperpolarising tyrosine catabolites dopamine, noradrenaline and morphine contributing to membrane Na +–K + ATPase inhibition in all the above disorders and the indexed family. Digoxin induced membrane Na +–K + ATPase inhibition can result in increased intracellular Ca 2+ and reduced Mg ++ levels leading to glutamate excitotoxicity, oncogene activation and immune activation. Digoxin induced altered Ca ++/Mg ++ ratios, reduced ubiquinone and increased dolichol can affect glycoconjugate metabolism, membrane formation and structure and mitochondrial function leading to the diverse disorders described above including those in the indexed family. The isoprenoid pathway and neurotransmitter patterns were compared in right-handed/left hemispheric dominant and left-handed/right hemispheric dominant individuals. The biochemical patterns in the indexed family and the diverse disorders studied correlated with those obtained in right hemispheric dominance. The hyperdigoxinemic state indicates right hemispheric dominance. Hypothalamic digoxin can thus function as the master conductor of the neuroimmunoendocrine orchestra and co-ordinate the functions of various cellular organelles.

A HYPOTHALAMIC DIGOXIN-MEDIATED MODEL FOR AUTISM

The isoprenoid pathway and its metabolites--digoxin, dolichol, and ubiquinon--were assessed in autism. The isoprenoid pathway and digoxin status was also studied for comparison in individuals of differing hemispheric dominance to determine the role of cerebral dominance in the genesis of autism. There was an upregulation of the isoprenoid pathway as evidenced by elevated HMG CoA reductase activity in autism. Digoxin, an endogenous Na+-K+ ATPase inhibitor secreted by the hypothalamus, was found to be elevated and RBC membrane Na+-K+ ATPase activity was found to be reduced in autism. Membrane Na+-K+ ATPase inhibition can result in increased intracellular Ca2+ and reduced magnesium levels. Hypothalamic digoxin can modulate conscious and subliminal perception and its dysfunction may lead to autism. Digoxin can also preferentially upregulate tryptophan transport over tyrosine resulting in increased levels of depolarizing tryptophan catabolites--serotonin, quinolinic acid (NMDA agonist), strychnine (blocks glycinergic inhibitory transmission), and nicotine (promotes dopamine release) and decreased levels of hyperpolarizing tyrosine catabolites--dopamine, noradrenaline, and morphine--contributing to membrane Na+-K+ ATPase inhibition. Increased nicotine levels can produce increased dopaminergic transmission in the presence of low dopamine levels. NMDA excitotoxicity could result from hypomagnesemia induced by membrane Na+K+ ATPase inhibition and quinolinic acid, an NMDA agonist acting on the NMDA receptor. Hypomagnesemia and increased dolichol level can affect glycoconjugate metabolism and membranogenesis leading on to disordered synaptic connectivity in the limbic allocortex and defective presentation of viral antigens and neuronal antigens contributing to autoimmunity and viral persistance important in the pathogenesis. Membrane Na+-K+ ATPase inhibition can produce immune activation, a component of autoimmunity. Mitochondrial dysfunction consequent to altered calcium/magnesium ratios and reduced ubiquinone levels can result in increased free radical generation and reduced free radical scavenging and defective apoptosis leading to abnormal synaptogenesis. Autism can thus be considered a syndrome of hypothalamic digoxin hypersecretion consequent to an upregulated isoprenoid pathway. The biochemical patterns including hyperdigoxinemia observed in autism correlated with those obtained in right hemispheric chemical dominance. Right hemispheric chemical dominance is a predisposing factor for autism

Annexin II light chain regulates sensory neuron-specific sodium channel expression.

The tetrodotoxin-resistant sodium channel Na(V)1.8/SNS is expressed exclusively in sensory neurons and appears to have an important role in pain pathways. Unlike other sodium channels, Na(V)1.8 is poorly expressed in cell lines even in the presence of accessory beta-subunits. Here we identify annexin II light chain (p11) as a regulatory factor that facilitates the expression of Na(V)1.8. p11 binds directly to the amino terminus of Na(V)1.8 and promotes the translocation of Na(V)1.8 to the plasma membrane, producing functional channels. The endogenous Na(V)1.8 current in sensory neurons is inhibited by antisense downregulation of p11 expression. Because direct association with p11 is required for functional expression of Na(V)1.8, disrupting this interaction may be a useful new approach to downregulating Na(V)1.8 and effecting analgesia.