Myo-Inositol-Dependent Sodium Uptake in Ice Plant1

In salt-stressed ice plants (Mesembryanthemum crystallinum), sodium accumulates to high concentrations in vacuoles, and polyols (myo-inositol, d-ononitol, and d-pinitol) accumulate in the cytosol. Polyol synthesis is regulated by NaCl and involves induction and repression of gene expression (D.E. Nelson, B. Shen, and H.J. Bohnert [1998] Plant Cell 10: 753–764). In the study reported here we found increased phloem transport of myo-inositol and reciprocal increased transport of sodium and inositol to leaves under stress. To determine the relationship between increased translocation and sodium uptake, we analyzed the effects of exogenous application of myo-inositol: The NaCl-inducible ice plant myo-inositol 1-phosphate synthase is repressed in roots, and sodium uptake from root to shoot increases without stimulating growth. Sodium uptake and transport through the xylem was coupled to a 10-fold increase of myo-inositol and ononitol in the xylem. Seedlings of the ice plant are not salt-tolerant, and yet the addition of exogenous myo-inositol conferred upon them patterns of gene expression and polyol accumulation observed in mature, salt-tolerant plants. Sodium uptake and transport through the xylem was enhanced in the presence of myo-inositol. The results indicate an interdependence of sodium uptake and alterations in the distribution of myo-inositol. We hypothesize that myo-inositol could serve not only as a substrate for the production of compatible solutes but also as a leaf-to-root signal that promotes sodium uptake.

Sodium channels and pain

Although it is well established that hyperexcitability and/or increased baseline sensitivity of primary sensory neurons can lead to abnormal burst activity associated with pain, the underlying molecular mechanisms are not fully understood. Early studies demonstrated that, after injury to their axons, neurons can display changes in excitability, suggesting increased sodium channel expression, and, in fact, abnormal sodium channel accumulation has been observed at the tips of injured axons. We have used an ensemble of molecular, electrophysiological, and pharmacological techniques to ask: what types of sodium channels underlie hyperexcitability of primary sensory neurons after injury? Our studies demonstrate that multiple sodium channels, with distinct electrophysiological properties, are encoded by distinct mRNAs within small dorsal root ganglion (DRG) neurons, which include nociceptive cells. Moreover, several DRG neuron-specific sodium channels now have been cloned and sequenced. After injury to the axons of DRG neurons, there is a dramatic change in sodium channel expression in these cells, with down-regulation of some sodium channel genes and up-regulation of another, previously silent sodium channel gene. This plasticity in sodium channel gene expression is accompanied by electrophysiological changes that poise these cells to fire spontaneously or at inappropriate high frequencies. Changes in sodium channel gene expression also are observed in experimental models of inflammatory pain. Thus, sodium channel expression in DRG neurons is dynamic, changing significantly after injury. Sodium channels within primary sensory neurons may play an important role in the pathophysiology of pain.

A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain

Alterations in sodium channel expression and function have been suggested as a key molecular event underlying the abnormal processing of pain after peripheral nerve or tissue injury. Although the relative contribution of individual sodium channel subtypes to this process is unclear, the biophysical properties of the tetrodotoxin-resistant current, mediated, at least in part, by the sodium channel PN3 (SNS), suggests that it may play a specialized, pathophysiological role in the sustained, repetitive firing of the peripheral neuron after injury. Moreover, this hypothesis is supported by evidence demonstrating that selective “knock-down” of PN3 protein in the dorsal root ganglion with specific antisense oligodeoxynucleotides prevents hyperalgesia and allodynia caused by either chronic nerve or tissue injury. In contrast, knock-down of NaN/SNS2 protein, a sodium channel that may be a second possible candidate for the tetrodotoxin-resistant current, appears to have no effect on nerve injury-induced behavioral responses. These data suggest that relief from chronic inflammatory or neuropathic pain might be achieved by selective blockade or inhibition of PN3 expression. In light of the restricted distribution of PN3 to sensory neurons, such an approach might offer effective pain relief without a significant side-effect liability.

Evolution and divergence of sodium channel genes in vertebrates

Invertebrate species possess one or two Na+ channel genes, yet there are 10 in mammals. When did this explosive growth come about during vertebrate evolution? All mammalian Na+ channel genes reside on four chromosomes. It has been suggested that this came about by multiple duplications of an ancestral chromosome with a single Na+ channel gene followed by tandem duplications of Na+ channel genes on some of these chromosomes. Because a large-scale expansion of the vertebrate genome likely occurred before the divergence of teleosts and tetrapods, we tested this hypothesis by cloning Na+ channel genes in a teleost fish. Using an approach designed to clone all of the Na+ channel genes in a genome, we found six Na+ channel genes. Phylogenetic comparisons show that each teleost gene is orthologous to a Na+ channel gene or gene cluster on a different mammalian chromosome, supporting the hypothesis that four Na+ channel genes were present in the ancestors of teleosts and tetrapods. Further duplications occurred independently in the teleost and tetrapod lineages, with a greater number of duplications in tetrapods. This pattern has implications for the evolution of function and specialization of Na+ channel genes in vertebrates. Sodium channel genes also are linked to homeobox (Hox) gene clusters in mammals. Using our phylogeny of Na+ channel genes to independently test between two models of Hox gene evolution, we support the hypothesis that Hox gene clusters evolved as (AB) (CD) rather than {D[A(BC)]}.

Antillatoxin is a marine cyanobacterial toxin that potently activates voltage-gated sodium channels

Antillatoxin (ATX) is a lipopeptide derived from the pantropical marine cyanobacterium Lyngbya majuscula. ATX is neurotoxic in primary cultures of rat cerebellar granule cells, and this neuronal death is prevented by either N-methyl-d-aspartate (NMDA) receptor antagonists or tetrodotoxin. To further explore the potential interaction of ATX with voltage-gated sodium channels, we assessed the influence of tetrodotoxin on ATX-induced Ca2+ influx in cerebellar granule cells. The rapid increase in intracellular Ca2+ produced by ATX (100 nM) was antagonized in a concentration-dependent manner by tetrodotoxin. Additional, more direct, evidence for an interaction with voltage-gated sodium channels was derived from the ATX-induced allosteric enhancement of [3H]batrachotoxin binding to neurotoxin site 2 of the α subunit of the sodium channel. ATX, moreover, produced a strong synergistic stimulation of [3H]batrachotoxin binding in combination with brevetoxin, which is a ligand for neurotoxin site 5 on the voltage-gated sodium channel. Positive allosteric interactions were not observed between ATX and either α-scorpion toxin or the pyrethroid deltamethrin. That ATX interaction with voltage-gated sodium channels produces a gain of function was demonstrated by the concentration-dependent and tetrodotoxin-sensitive stimulation of 22Na+ influx in cerebellar granule cells exposed to ATX. Together these results demonstrate that the lipopeptide ATX is an activator of voltage-gated sodium channels. The neurotoxic actions of ATX therefore resemble those of brevetoxins that produce neural insult through depolarization-evoked Na+ load, glutamate release, relief of Mg2+ block of NMDA receptors, and Ca2 + influx.

Cyanogenesis in Cassava1 : The Role of Hydroxynitrile Lyase in Root Cyanide Production

In the cyanogenic crop cassava (Manihot esculenta, Crantz), the final step in cyanide production is the conversion of acetone cyanohydrin, the deglycosylation product of linamarin, to cyanide plus acetone. This process occurs spontaneously at pH greater than 5.0 or enzymatically and is catalyzed by hydroxynitrile lyase (HNL). Recently, it has been demonstrated that acetone cyanohydrin is present in poorly processed cassava root food products. Since it has generally been assumed that HNL is present in all cassava tissues, we reinvestigated the enzymatic properties and tissue-specific distribution of HNL in cassava. We report the development of a rapid two-step purification protocol for cassava HNL, which yields an enzyme that is catalytically more efficient than previously reported (Hughes, J., Carvalho, F., and Hughes, M. [1994] Arch Biochem Biophys 311: 496–502). Analyses of the distribution of HNL activity and protein indicate that the accumulation of acetone cyanohydrin in roots is due to the absence of HNL, not to inhibition of the enzyme. Furthermore, the absence of HNL in roots and stems is associated with very low steady-state HNL transcript levels. It is proposed that the lack of HNL in cassava roots accounts for the high acetone cyanohydrin levels in poorly processed cassava food products.

Polyunsaturated fatty acids modulate sodium and calcium currents in CA1 neurons.

Recent evidence indicates that long-chain polyunsaturated fatty acids (PUFAs) can prevent cardiac arrhythmias by a reduction of cardiomyocyte excitability. This was shown to be due to a modulation of the voltage-dependent inactivation of both sodium (INa) and calcium (ICa) currents. To establish whether PUFAs also regulate neuronal excitability, the effects of PUFAs on INa and ICa were assessed in CA1 neurons freshly isolated from the rat hippocampus. Extracellular application of PUFAs produced a concentration-dependent shift of the voltage dependence of inactivation of both INa and ICa to more hyperpolarized potentials. Consequently, they accelerated the inactivation and retarded the recovery from inactivation. The EC50 for the shift of the INa steady-state inactivation curve was 2.1 +/- 0.4 microM for docosahexaenoic acid (DHA) and 4 +/- 0.4 microM for eicosapentaenoic acid (EPA). The EC50 for the shift on the ICa inactivation curve was 2.1 +/- 0.4 for DHA and > 15 microM for EPA. Additionally, DHA and EPA suppressed both INa and ICa amplitude at concentrations > 10 microM. PUFAs did not affect the voltage dependence of activation. The monounsaturated oleic acid and the saturated palmitic acid were virtually ineffective. The combined effects of the PUFAs on INa and ICa may reduce neuronal excitability and may exert anticonvulsive effects in vivo.

Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.

The pores of voltage-gated ion channels are lined by protein loops that determine selectivity and conductance. The relative orientations of these "P" loops remain uncertain, as do the distances between them. Using site-directed mutagenesis, we introduced pairs of cysteines into the P loops of micro1 rat skeletal muscle sodium channels and sought functional evidence of proximity between the substituted residues. Only cysteinyl residues that are in close proximity can form disulfide bonds or metal-chelating sites. The mutant Y401C (domain I) spontaneously formed a disulfide bond when paired with E758C in the P loop of domain II; the same residue, when coupled with G1530C in domain IV, created a high-affinity binding site for Cd2+ ions. The results provide the first specific constraints for intramolecular dimensions of the sodium channel pore.

The heart communicates with the kidney exclusively through the guanylyl cyclase-A receptor: acute handling of sodium and water in response to volume expansion.

Disruption of guanylyl cyclase-A (GC-A) results in mice displaying an elevated blood pressure, which is not altered by high or low dietary salt. However, atrial natriuretic peptide (ANP), a proposed ligand for GC-A, has been suggested as critical for the maintenance of normal blood pressure during high salt intake. In this report, we show that infusion of ANP results in substantial natriuresis and diuresis in wild-type mice but fails to cause significant changes in sodium excretion or urine output in GC-A-deficient mice. ANP, therefore, appears to signal through GC-A in the kidney. Other natriuretic/diuretic factors could be released from the heart. Therefore, acute volume expansion was used as a means to cause release of granules from the atrium of the heart. That granule release occurred was confirmed by measurements of plasma ANP concentrations, which were markedly elevated in both wild-type and GC-A-null mice. After volume expansion, urine output as well as urinary sodium and cyclic GMP excretion increased rapidly and markedly in wild-type mice, but the rapid increases were abolished in GC-A-deficient animals. These results strongly suggest that natriuretic/diuretic factors released from the heart function exclusively through GC-A.

Expression of a renal type I sodium/phosphate transporter (NaPi-1) induces a conductance in Xenopus oocytes permeable for organic and inorganic anions.

Two distinct molecular types (I and II) of renal proximal tubular brush border Na+/Pi cotransporters have been identified by expression cloning on the basis of their capacity to induce Na+-dependent Pi influx in tracer experiments. Whereas the type II transporters (e.g., NaPi-2 and NaPi-3) resemble well known characteristics of brush border Na+/Pi cotransport, little is known about the properties of the type I transporter (NaPi-1). In contrast to type II, type I transporters produced electrogenic transport only at high extracellular Pi concentrations (> or =3 mM). On the other hand, expression of NaPi-1 induced a Cl- conductance in Xenopus laevis oocytes, which was inhibited by Cl- channel blockers [5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) > niflumic acid >> 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid]. Further, the Cl- conductance was inhibited by the organic anions phenol red, benzylpenicillin (penicillin G), and probenecid. These organic anions induced outwardly directed currents in the absence of Cl-. In tracer studies, we observed uptake of benzylpenicillin with a Km of 0.22 mM; benzylpenicillin uptake was inhibited by NPPB and niflumic acid. These findings suggest that the type I Na+/Pi cotransporter functions also as a novel type of anion channel permeable not only for Cl- but also for organic anions. Such an apical anion channel could serve an important role in the transport of Cl- and the excretion of anionic xenobiotics.

Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists.

Na+-Ca2+ exchanger and Ca2+ channel are two major sarcolemmal Ca2+-transporting proteins of cardiac myocytes. Although the Ca2+ channel is effectively regulated by protein kinase A-dependent phosphorylation, no enzymatic regulation of the exchanger protein has been identified as yet. Here we report that in frog ventricular myocytes, isoproterenol down-regulates the Na+-Ca2+ exchanger, independent of intracellular Ca2+ and membrane potential, by activation of the beta-receptor/adenylate-cyclase/cAMP-dependent cascade, resulting in suppression of transmembrane Ca2+ transport via the exchanger and providing for the well-documented contracture-suppressant effect of the hormone on frog heart. The beta-blocker propranolol blocks the isoproterenol effect, whereas forskolin, cAMP, and theophylline mimic it. In the frog heart where contractile Ca2+ is transported primarily by the Na+-Ca2+ exchanger, the beta-agonists' simultaneous enhancement of Ca2+ current, ICa, and suppression of Na+-Ca2+ exchanger current, INa-Ca would enable the myocyte to develop force rapidly at the onset of depolarization (enhancement of ICa) and to decrease Ca2+ influx (suppression of INa-Ca) later in the action potential. This unique adrenergically induced shift in the Ca2+ influx pathways may have evolved in response to paucity of the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex and absence of significant intracellular Ca2+ release pools in the frog heart.

Synergy between chronic corticosterone and sodium azide treatments in producing a spatial learning deficit and inhibiting cytochrome oxidase activity.

Previously, we developed a rat model of persistent mitochondrial dysfunction based upon the chronic partial inhibition of the mitochondrial enzyme cytochrome oxidase (EC 1.9.3.1). Continuous systemic infusion of sodium azide at approximately 1 mg/kg per hr inhibited cytochrome oxidase activity and produced a spatial learning deficit. In other laboratories, glucocorticoids have been reported to exacerbate neuronal damage from various acute metabolic insults. Therefore, we tested the hypothesis that corticosterone, the primary glucocorticoid in the rat, would potentiate the sodium azide-induced learning deficit. To this end, we first identified nonimpairing doses of sodium azide (approximately 0.75 mg/kg per hr) and corticosterone (100-mg pellet, 3-week sustained-release). We now report that chronic co-administration of these individually nonimpairing treatments produced a severe learning deficit. Moreover, the low dose of corticosterone, which did not elevate serum corticosterone, acted synergistically with sodium azide to inhibit cytochrome oxidase activity. The latter result represents a previously unidentified effect of glucocorticoids that provides a candidate mechanism for glucocorticoid potentiation of neurotoxicity induced by metabolic insult. These results may have the clinical implication of expanding the definition of hypercortisolism in patient populations with compromised oxidative metabolism. Furthermore, they suggest that glucocorticoid treatment may contribute to pathology in disease or trauma conditions that involve metabolic insult.

Sodium: a male moth's gift to its offspring.

Males of the moth Gluphisia septentrionis acquire sodium by drinking from mud puddles. Analyses of male and female bodies indicate that such "puddling" behavior enables the male to provide his mate with a nuptial gift of sodium, presumably via the spermatophore. This gift (about 10 microg), amounting to more than half of a puddler male's total body sodium, is in large measure apportioned by the female to her eggs. Puddler-sired eggs contain 2 to 4 times more sodium than those control-sired; this difference is already apparent in eggs laid the night after mating. Paternal endowment of offspring with sodium had not previously been demonstrated for an insect to our knowledge. The potential adaptive significance of such chemical bestowal is evident, given that the foliar diet of G. septentrionis larvae is extremely low in sodium content.