Glutamate Uptake Stimulates Na+,K+-ATPase Activity in Astrocytes via Activation of a Distinct Subunit Highly Sensitive to Ouabain

Abstract: The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na⁺-dependent transporter. Evidence had been provided that Na⁺,K⁺-ATPase might be involved in this process. We have now measured the activity of Na⁺,K⁺-ATPase in cultured astrocytes, using ouabain-sensitive ⁸⁶Rb uptake as an index. l -Glutamate increases glial Na⁺,K⁺-ATPase activity in a concentration-dependent manner with an EC₅₀ = 67 µ M . Both l - and d -aspartate, but not d -glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na⁺,K⁺-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain ( K _0.5 = 113 µ M ), compatible with the presence of an α₁ isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain ( K _0.5 = 20 n M ), thus revealing a high-affinity site akin to the α₂ isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na⁺,K⁺-ATPase that can be mobilized in response to increased neuronal activity.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

Isoform-Specific Up-Regulation by Ouabain of Na+,K+-ATPase in Cultured Rat Astrocytes

Abstract: There are two α-subunit isoforms (α1 and α2) and two β-subunit isoforms (β1 and β2) of Na⁺,K⁺-ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5–1.0 m M ) increased the levels of α1 and β1 mRNAs, whereas it decreased those of α2 and β2 mRNAs in cultured rat astrocytes. The increases in α1 and β1 mRNAs were observed at 6–48 h after addition of the inhibitor. Immunochemical analyses showed that ouabain increased α1 and β1, but not α2 and β2, proteins, and that the isoforms in control and ouabain-treated cultures were of glial origin. Low extracellular K⁺ and monensin (20 µ M ) mimicked the effect of ouabain on α1 mRNA. The ouabain-induced increase in α1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 µ M ), the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N',N' -tetraacetic acid tetraacetoxymethyl ester (30 µ M ), and the calcineurin inhibitor FK506 (1 n M ). These findings indicate that chronic inhibition of Na⁺,K⁺-ATPase up-regulates the α1 and β1, but not α2 and β2, isoforms in astrocytes, suggesting a functional coupling of α1β1 complex. They also suggest that intracellular Na⁺, Ca²⁺, and calcineurin may be involved in ouabain-induced up-regulation of the enzyme in astrocytes.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Mechanisms of Sodium Transport at the Blood-Brain Barrier Studied with In Situ Perfusion of Rat Brain

Abstract: The mechanism of unidirectional transport of sodium from blood to brain in pentobarbital-anesthetized rats was examined using in situ perfusion. Sodium transport followed Michaelis-Menten saturation kinetics with a V _max of 50.1 nmol/g/min and a K _m of 17.7 m M in the left frontal cortex. The kinetic analysis indicated that, at a physiologic sodium concentration, ∼26% of sodium transport at the blood-brain barrier (BBB) was carrier mediated. Dimethylamiloride (25 µ M ), an inhibitor of Na⁺/H⁺ exchange, reduced sodium transport by 28%, whereas phenamil (25 µ M ), a sodium channel inhibitor, reduced the transfer constant for sodium by 22%. Bumetanide (250 µ M ) and hydrochlorothiazide (1.5 m M ), inhibitors of Na⁺-K⁺-2Cl⁻/NaCl symport, were ineffective in reducing blood to brain sodium transport. Acetazolamide (0.25 m M ), an inhibitor of carbonic anhydrase, did not change sodium transport at the BBB. Finally, a perfusate pH of 7.0 or 7.8 or a perfusate P co ₂ of 86 mm Hg failed to change sodium transport. These results indicate that 50% of transcellular transport of sodium from blood to brain occurs through Na⁺/H⁺ exchange and a sodium channel in the luminal membrane of the BBB. We propose that the sodium transport systems at the luminal membrane of the BBB, in conjunction with Cl⁻/HCO₃⁻ exchange, lead to net NaCl secretion and obligate water transport into the brain.     

Modulation of Human Glutamate Transporter Activity by Phorbol Ester

Abstract: Termination of synaptic glutamate transmission depends on rapid removal of glutamate by neuronal and glial high-affinity transporters. Molecular biological and pharmacological studies have demonstrated that at least five subtypes of Na⁺-dependent mammalian glutamate transporters exist. Our study demonstrates that Y-79 human retinoblastoma cells express a single Na⁺-dependent glutamate uptake system with a K _m of 1.7 ± 0.42 µ M that is inhibited by dihydrokainate and dl - threo -β-hydroxyaspartate (IC₅₀ = 0.29 ± 0.17 µ M and 2.0 ± 0.43 µ M , respectively). The protein kinase C activator phorbol 12-myristate 13-acetate caused a concentration-dependent inhibition of glutamate uptake (IC₅₀ = 0.56 ± 0.05 n M ), but did not affect Na⁺-dependent glycine uptake significantly. This inhibition of glutamate uptake resulted from a fivefold decrease in the transporter's affinity for glutamate, without significantly altering the V _max. 4α-Phorbol 12,13-didecanoate, a phorbol ester that does not activate protein kinase C, did not alter glutamate uptake significantly. The phorbol 12-myristate 13-acetate-induced inhibition of glutamate uptake was reversed by preincubation with staurosporine. The biophysical and pharmacological profile of the human glutamate transporter expressed by the Y-79 cell line indicates that it belongs to the dihydrokainate-sensitive EAAT2/GLT-1 subtype. This conclusion was confirmed by western blot analysis. Protein kinase C modulation of glutamate transporter activity may represent a mechanism to modulate extracellular glutamate and shape postsynaptic responses.     

Inhibition of Depolarization-Induced Nitric Oxide Synthase Activation by NS-7, a Phenylpyrimidine Derivative, in Primary Neuronal Culture

Abstract: Neuronal nitric oxide synthase (NOS) is considered to be involved in the pathogenesis of ischemic brain damage. In the present study, the effect of a novel neuroprotective phenylpyrimidine derivative, 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride (NS-7), on depolarization-stimulated NOS activity was examined in cultured neurons of mouse cerebral cortex. Various depolarizing stimuli such as veratridine, KCl, and N -methyl- d -aspartate increased the NOS activity determined by cyclic GMP formation. NS-7 concentration-dependently inhibited both the veratridine- and KCl-induced NOS activation with IC₅₀ values of 9.3 and 9.6 µ M , respectively. The reversal of KCl-evoked NOS activity by NS-7 was also observed under blockade of both ionotropic glutamate receptors and the Na⁺ channel with MK-801, 6-cyano-7-nitroquinoxaline-2,3-dione, and tetrodotoxin. In contrast, NS-7, even at 100 µ M , did not affect N -methyl- d -aspartate-stimulated NOS activity, nor did it have any influence on NOS activity determined in the soluble fraction of rat hippocampus. Because NS-7 has already been shown to block both Na⁺ and Ca²⁺ channels, the present findings suggest that this compound inhibits depolarization-induced NOS activation by reducing Ca²⁺ influx through blockade of Na⁺ and Ca²⁺ channels in primary neuronal culture.     

Heterogeneous Na+ Sensitivity of Na+,K+-ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na⁺ sensitivity of whole brain membrane Na⁺,K⁺-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 m M Na⁺, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10⁻⁵ and 10⁻⁷ M ), we were able to discriminate Na⁺ sensitivity of Na⁺, K⁺-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, α1, exhibited high Na⁺ sensitivity [ K _a of 3.88 ± 0.25 m M Na⁺ and a Hill coefficient ( n ) of 1.98 ± 0.13]; the ouabain high-affinity isoform, α2, had two Na⁺ sensitivities, a high ( K _a of 4.98 ± 0.2 m M Na⁺ and n of 1.34 ± 0.10) and a low ( K _a of 28 ± 0.5 m M Na⁺ and an n of 1.92 ± 0.18) Na⁺ sensitivity activated above a thresh old (22 ± 0.3 m M Na⁺); and the ouabain very-high-affinity isoform, α3, was resolved by two processes and appears to have two Na⁺ sensitivities (apparent K _a values of 3.5 and 20 m M Na⁺). We show that Na⁺ dependence in the absence of ouabain is the result of at least of five Na⁺ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

Glutamate Induces a Calcineurin-Mediated Dephosphorylation of Na+,K+-ATPase that Results in Its Activation in Cerebellar Neurons in Culture

Abstract: In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca²⁺ and Na⁺ into the neuron. To maintain Na⁺ homeostasis, the excess Na⁺ entering through the ion channel should be removed by Na⁺,K⁺-ATPase. It is shown that incubation of primary cultured cerebellar neurons with glutamate resulted in activation of the Na⁺,K⁺-ATPase. The effect was rapid, peaking between 5 and 15 min (85% activation), and was maintained for at least 2 h. Glutamate-induced activation of Na⁺,K⁺-ATPase was dose dependent: It was appreciable (37%) at 0.1 µ M and peaked (85%) at 100 µ M . The increase in Na⁺,K⁺-ATPase activity by glutamate was prevented by MK-801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase was reversed by phorbol 12-myristate 13-acetate, an activator of protein kinase C, indicating that activation of Na⁺,K⁺-ATPase is due to decreased phosphorylation by protein kinase C. W-7 or cyclosporin, both inhibitors of calcineurin, prevented the activation of Na⁺,K⁺-ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na⁺,K⁺-ATPase that was previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na⁺,K⁺-ATPase.     

Nitroprusside and Cyclic GMP Stimulate Na+-Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes

Abstract: The effects of nitric oxide (NO)-generating agents on ⁴⁵Ca²⁺ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca²⁺ uptake in the slices, although it did not affect high K⁺-stimulated Ca²⁺ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca²⁺ uptake, but it did not affect high K⁺-stimulated Ca²⁺ uptake. 8-Bromocyclic GMP, but not SNP, increased Na⁺-dependent Ca²⁺ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca²⁺ uptake in the presence of ouabain and monensin, which were required for the Ca²⁺ uptake in the cells. These findings suggest that NO stimulates the Na⁺-Ca²⁺ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.     

Differential Inhibition of Secretagogue-Stimulated Sodium Uptake in Adrenal Chromaffin Cells by Activation of D4 and D5 Dopamine Receptors

Abstract: Recent studies have demonstrated that D1-selective and D2-selective dopamine receptor agonists inhibit catecholamine secretion and Ca²⁺ uptake into bovine adrenal chromaffin cells by receptor subtypes that we have identified by PCR as D5, a member of the D1-like dopamine receptor subfamily, and D4, a member of the D2-like dopamine receptor subfamily. The purpose of this study was to determine whether activation of D5 or D4 receptors inhibits influx of Na⁺, which could explain inhibition of secretion and Ca²⁺ uptake by dopamine agonists. D1-selective agonists preferentially inhibited both dimethylphenylpiperazinium- (DMPP) and veratridine-stimulated ²²Na⁺ influx into chromaffin cells. The D1-selective agonists chloro-APB hydrobromide (CI-APB; 100 µ M ) and SKF-38393 (100 µ M ) inhibited DMPP-stimulated Na⁺ uptake by 87.5 ± 2.3 and 59.7 ± 4.5%, respectively, whereas the D2-selective agonist bromocriptine (100 µ M ) inhibited Na⁺ uptake by only 22.9 ± 5.0%. Veratridine-stimulated Na⁺ uptake was inhibited 95.1 ± 3.2 and 25.7 ± 4.7% by 100 µ M CI-APB or bromocriptine, respectively. The effect of CI-APB was concentration dependent. A similar IC₅₀ (∼18 µ M ) for inhibition of both DMPP- and veratridine-stimulated Na⁺ uptake was obtained. The addition of 8-bromo-cyclic AMP (1 m M ) had no effect on either DMPP- or veratridine-stimulated Na⁺ uptake. These observations suggest that D1-selective agonists are inhibiting secretagogue-stimulated Na⁺ uptake in a cyclic AMP-independent manner.     

NMDA Receptor-Mediated Neurotoxicity: A Paradoxical Requirement for Extracellular Mg2+ in Na+/Ca2+-Free Solutions in Rat Cortical Neurons In Vitro

Abstract: Accumulation of intracellular Ca²⁺ is known to be critically important for the expression of NMDA receptor-mediated glutamate neurotoxicity. We have observed, however, that glutamate can also increase the neuronal intracellular Mg²⁺ concentration on activation of NMDA receptors. Here, we used conditions that elevate intracellular Mg²⁺ content independently of Ca²⁺ to investigate the potential role of Mg²⁺ in excitotoxicity in rat cortical neurons in vitro. In Ca²⁺-free solutions in which the Na⁺ was replaced by N -methyl- d -glucamine or Tris (but not choline), which also contained 9 m M Mg²⁺, exposure to 100 µ M glutamate or 200 µ M NMDA for 20 min produced delayed neuronal cell death. Neurotoxicity was correlated to the extracellular Mg²⁺ concentration and could be blocked by addition of NMDA receptor antagonists during, but not immediately following, agonist exposure. Finally, we observed that rat cortical neurons grown under different serum conditions develop an altered sensitivity to Mg²⁺-dependent NMDA receptor-mediated toxicity. Thus, the increase in intracellular Mg²⁺ concentration following NMDA receptor stimulation may be an underestimated component critical for the expression of certain forms of excitotoxic injury.     

Glutamine Transport in Mouse Cerebral Astrocytes

Abstract: We measured initial influx and exchange of [¹⁴C]glutamine in primary astrocyte cultures in the presence and absence of Na⁺. Kinetic analysis of transport in Na⁺-free solution indicated two saturable Na⁺-independent components, one of which was identifiable functionally as system L₁ transport. In the presence of Na⁺, multiple hyperbolic components were not resolvable from the kinetic data. Nevertheless, other evidence supported participation by at least three Na⁺-dependent neutral amino acid transporters (systems A, ASC, and N). System A transport of glutamine was usually absent or minimal, based on lack of inhibition by α-(methylamino)isobutyric acid. However, vigorous system A-mediated transport emerged after derepression by substrate deprivation. Participation by system ASC was indicated by trans-acceleration of Na⁺-dependent uptake, preferential inhibition of an Li⁺-intolerant component of uptake by cysteine, and inhibition by cysteine of a component resistant to inhibition by histidine and α-(methylamino)isobutyric acid. Because nonsaturable transport of glutamine appeared negligible, and system L transport of glutamine was suppressed in the presence of Na⁺, low-affinity system ASC transport may be the major route of export of glutamine from astrocytes. At 700 µ M glutamine, the primary uptake route was system N transport, identified on the basis of selective inhibition by histidine and asparagine, pH sensitivity, and tolerance of Li⁺ in place of Na⁺.     

Effect of Fish Oil Diet on Fatty Acid Composition of Phospholipids of Brain Membranes and on Kinetic Properties of Na+, K+-ATPase Isoenzymes of Weaned and Adult Rats

Abstract: The influence of dietary (n-3) fatty acids (such as eicosapentaenoic and docosahexaenoic acids) as found in fish oil on Na⁺ sensitivity and ouabain affinity of Na⁺, K⁺-ATPase isoenzymes (α1, α2, α3) was studied in whole brain membranes from weaned and adult rats fed diets for two generations. The long chain (n-3) fatty acids supplied by fish oil decreased the fatty acids of the (n-6) series compared with the standard diet, resulting in a decrease in the (n-6)/(n-3) molar ratio in both 21 - and 60-day- old rats. On the basis of ouabain titration, three inhibitory processes with markedly different affinities were associated with isoenzymes, i.e., low affinity (α1), high affinity (α2), and very high affinity (α3). It appears that the fish oil diet, in part via the modification of membrane fatty acid composition, altered the proportion and ouabain affinity of isoenzymes. Na⁺ sensitivity is the best criterion of physiologic change induced by fish oil diet. We calculated the Na⁺ activation for each isoenzyme and found one Na⁺ sensitivity and two Na⁺ sensitivities per isoenzyme in weanling and adult rats fed different diets, respectively. In contrast to α2 and α3, α1 appears insensitive to membrane change induced by fish oil diet. Fish oil diet, which is known to confer cardioprotection, induced significant modulation of Na⁺, K⁺-ATPase isoenzymes at the brain level.     

K+ status and ABA affect both exudation rate and hydraulic conductivity in sunflower roots

Twenty‐day‐old sunflower plants ( Helianthus annuus L. cv. Sun‐Gro 380) grown in nutrient solutions with different KCl levels were used to study the effects of K⁺ status of the root and of abcisic acid (ABA) on the exudation rate (J_v), the hydraulic conductivity of the root (L_p), the fluxes of exuded K⁺ and Na⁺ (J_K and J_Na), and the gradient of osmotic pressure between the xylem and the external medium. J_v and L_p increased in direct proportion to the K⁺ starvation of the root. Also addition of ABA (4 µ M ) at the onset of exudation in the external medium made J_v and L_p rise, and this effect also increased with the degree of K⁺ starvation. Similarly, K⁺ starvation and ABA promoted both the flux of exuded Na⁺ and the accumulation of Na⁺ in the root. We suggest that ABA acts as a regulating signal for the radial transport of water across the root, and that potassium may be an effector of this mechanism.     

Detection of Intracellular Free Na+ Concentration of Synaptosomes by a Fluorescent Indicator, Na+-Binding Benzofuran Isophthalate: The Effect of Veratridine, Ouabain, and α-Latrotoxin

Abstract: A novel fluorescent Na⁺ indicator, Na⁺-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na⁺ concentration ([Na⁺]₁) of synaptosomes. The dye, when loaded into synapto- somes in the form of its acetoxymethyl ester, was responsive to changes of [Na⁺]₁. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na⁺ concentration was equilibrated with different concentrations of extracellular Na⁺ in the presence of 2 μ M gramicidin D. The basal value of [Na⁺]₁ in synaptosomes in the presence of 140 m M extracellular Na⁺ was found to be 10.9 ± 1.8 m M. Veratridine, which opens potential-dependent Na⁺ channels, caused a sudden increase in [Na⁺]₁ in a concentration-dependent manner (1 -20 μ M ), whereas the effect of ouabain (20 and 50 μ M ), the inhibitor of the plasma membrane Na⁺,K⁺-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 μ M tetrodotoxin. α-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19–1.5 n M ). This report confirms our earlier finding demonstrating a Na⁺-dependent component in the action of α-Iatrotoxin, and shows that changes in [Na⁺]₁ in synaptosomes can be followed by SBFI.     

Axonal Contact Regulates Expression of α2 and β2 Isoforms of Na+,K+-ATPase in Schwann Cells: Adhesion Molecules and Nerve Regeneration

Abstract: Three isoforms of catalytic α subunits and two isoforms of β subunits of Na⁺,K⁺-ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na⁺,K⁺-ATPase was highly resistant to ouabain. The ouabain-resistant α1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na⁺,K⁺-ATPase activity. After sciatic nerve injury, the α3 and β1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, α2 and β2 isoform expression and Na⁺,K⁺-ATPase activity sensitive to pyrithiamine (a specific inhibitor of the α2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that α3 and β1 isoforms are exclusive for the axon and α2 and β2 isoforms are exclusive for the Schwann cell, although axonal contact regulates α2 and β2 isoform expressions. Because the β2 isoform of Na⁺,K⁺-ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/β2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/β2 may act as an adhesion molecule in peripheral nerve regeneration.