Function and Regulation of the Na+-Ca2+ Exchanger NCX3 Splice Variants in Brain and Skeletal Muscle

Isoform 3 of the Na⁺-Ca²⁺ exchanger (NCX3) is crucial for maintaining intracellular calcium ([Ca²⁺]_i) homeostasis in excitable tissues. In this sense NCX3 plays a key role in neuronal excitotoxicity and Ca²⁺ extrusion during skeletal muscle relaxation. Alternative splicing generates two variants (NCX3-AC and NCX3-B). Here, we demonstrated that NCX3 variants display a tissue-specific distribution in mice, with NCX3-B as mostly expressed in brain and NCX-AC as predominant in skeletal muscle. Using Fura-2-based Ca²⁺ imaging, we measured the capacity and regulation of the two variants during Ca²⁺ extrusion and uptake in different conditions. Functional studies revealed that, although both variants are activated by intracellular sodium ([Na⁺]_i), NCX3-AC has a higher [Na⁺]_i sensitivity, as Ca²⁺ influx is observed in the presence of extracellular Na⁺. This effect could be partially mimicked for NCX3-B by mutating several glutamate residues in its cytoplasmic loop. In addition, NCX3-AC displayed a higher capacity of both Ca²⁺ extrusion and uptake compared with NCX3-B, together with an increased sensitivity to intracellular Ca²⁺. Strikingly, substitution of Glu⁵⁸⁰ in NCX3-B with its NCX3-AC equivalent Lys⁵⁸⁰ recapitulated the functional properties of NCX3-AC regarding Ca²⁺ sensitivity, Lys⁵⁸⁰ presumably acting through a structure stabilization of the Ca²⁺ binding site. The higher Ca²⁺ uptake capacity of NCX3-AC compared with NCX3-B is in line with the necessity to restore Ca²⁺ levels in the sarcoplasmic reticulum during prolonged exercise. The latter result, consistent with the high expression in the slow-twitch muscle, suggests that this variant may contribute to the Ca²⁺ handling beyond that of extruding Ca²⁺.     

A mathematical model of Ca2+ dynamics in rat mesenteric smooth muscle cell: agonist and NO stimulation

A mathematical model of calcium dynamics in vascular smooth muscle cell (SMC) was developed based on data mostly from rat mesenteric arterioles. The model focuses on (a) the plasma membrane electrophysiology; (b) Ca²⁺ uptake and release from the sarcoplasmic reticulum (SR); (c) cytosolic balance of Ca²⁺, Na⁺, K⁺, and Cl⁻ ions; and (d) IP₃ and cGMP formation in response to norepinephrine (NE) and nitric oxide (NO) stimulation. Stimulation with NE induced membrane depolarization and an intracellular Ca²⁺ ([Ca²⁺]_i) transient followed by a plateau. The plateau concentrations were mostly determined by the activation of voltage-operated Ca²⁺ channels. NE causes a greater increase in [Ca²⁺]_i than stimulation with KCl to equivalent depolarization. Model simulations suggest that the effect of [Na⁺]_i accumulation on the Na⁺/Ca²⁺ exchanger (NCX) can potentially account for this difference. Elevation of [Ca²⁺]_i within a concentration window (150-300 nM) by NE or KCl initiated [Ca²⁺]_i oscillations with a concentration-dependent period. The oscillations were generated by the nonlinear dynamics of Ca²⁺ release and refilling in the SR. NO repolarized the NE-stimulated SMC and restored low [Ca²⁺]_i mainly through its effect on Ca²⁺-activated K⁺ channels. Under certain conditions, Na⁺-K⁺-ATPase inhibition can result in the elevation of [Na⁺]_i and the reversal of NCX, increasing resting cytosolic and SR Ca²⁺ content, as well as reactivity to NE. Blockade of the NCX's reverse mode could eliminate these effects. We conclude that the integration of the selected cellular components yields a mathematical model that reproduces, satisfactorily, some of the established features of SMC physiology. Simulations suggest a potential role of intracellular Na⁺ in modulating Ca²⁺ dynamics and provide insights into the mechanisms of SMC constriction, relaxation, and the phenomenon of vasomotion. The model will provide the basis for the development of multi-cellular mathematical models that will investigate microcirculatory function in health and disease.     

Ca2+ Influx via the Na+/Ca2+ Exchanger Is Enhanced in Malignant Hyperthermia Skeletal Muscle

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca²⁺ dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca²⁺ depending on cellular activity. Resting intracellular calcium ([Ca²⁺]_r) and sodium ([Na⁺]_r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering [Na⁺]_e elevates [Ca²⁺]_r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca²⁺ or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca²⁺]_r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca²⁺]_r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca²⁺]_r in MHS muscle fibers and decreases the amplitude of [Ca²⁺]_r rise triggered by halothane, but had no effect on [Ca²⁺]_r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca²⁺ transient elicited by high [K⁺]_e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca²⁺]_r and the Ca²⁺ transient area induced by high [K⁺]_e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca²⁺ transients associated with K⁺-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.     

Plasmalemmal Na+/Ca2+ exchanger modulates Ca2+-dependent exocytotic release of glutamate from rat cortical astrocytes

Astroglial excitability operates through increases in Ca²⁺_cyt (cytosolic Ca²⁺), which can lead to glutamatergic gliotransmission. In parallel fluctuations in astrocytic Na⁺_cyt (cytosolic Na⁺) control metabolic neuronal-glial signalling, most notably through stimulation of lactate production, which on release from astrocytes can be taken up and utilized by nearby neurons, a process referred to as lactate shuttle. Both gliotransmission and lactate shuttle play a role in modulation of synaptic transmission and plasticity. Consequently, we studied the role of the PMCA (plasma membrane Ca²⁺-ATPase), NCX (plasma membrane Na⁺/Ca²⁺ exchanger) and NKA (Na⁺/K⁺-ATPase) in complex and coordinated regulation of Ca²⁺_cyt and Na⁺_cyt in astrocytes at rest and upon mechanical stimulation. Our data support the notion that NKA and PMCA are the major Na⁺ and Ca²⁺ extruders in resting astrocytes. Surprisingly, the blockade of NKA or PMCA appeared less important during times of Ca²⁺ and Na⁺ cytosolic loads caused by mechanical stimulation. Unexpectedly, NCX in reverse mode appeared as a major contributor to overall Ca²⁺ and Na⁺ homoeostasis in astrocytes both at rest and when these glial cells were mechanically stimulated. In addition, NCX facilitated mechanically induced Ca²⁺-dependent exocytotic release of glutamate from astrocytes. These findings help better understanding of astrocyte-neuron bidirectional signalling at the tripartite synapse and/or microvasculature. We propose that NCX operating in reverse mode could be involved in fast and spatially localized Ca²⁺-dependent gliotransmission, that would operate in parallel to a slower and more widely distributed gliotransmission pathway that requires metabotropically controlled Ca²⁺ release from the ER (endoplasmic reticulum).     

Effect of α-Latrotoxin on Acetylcholine Release and Intracellular Ca2+ Concentration in Synaptosomes: Na+-Dependent and Na+-Independent Components

Abstract: We studied the effect of α-latrotoxin (αLTX) on [¹⁴C]acetylcholine ([¹⁴C]ACh) release, intracellular Ca²⁺ concentration ([Ca²⁺]_i), plasma membrane potential, and high-affinity choline uptake of synaptosomes isolated from guinea pig cortex. αLTX (10^?10-10^?8M) caused an elevation of the [Ca²⁺]_i as detected by Fura 2 fluorescence and evoked [¹⁴C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na⁺ in the external medium and another that did not. Displacement of Na⁺ by sucrose or N-methylglucamine in the medium considerably decreased the elevation of [Ca²⁺]_i and [¹⁴C]ACh release by αLTX. The Na⁺-dependent component of the αLTX action was obvious in the inhibition of the high-affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca²⁺]_i and [¹⁴C]ACh release remained in the absence of Na⁺. Both the Na⁺-dependent and the Na⁺-independent components of the αLTX-evoked [¹⁴C]ACh release partly required the presence of either Mg²⁺ or Ca²⁺. The nonneurotransmitter [¹⁴C]choline was released along with [¹⁴C]ACh, but this release did not depend on the presence of either Na⁺ or Ca²⁺, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation-dependent ACh release, which is related to (a) Na⁺-dependent divalent cation entry and (b) Na⁺-independent divalent cation entry, and (2) nonspecific Na⁺- and divalent cation-independent leakage.     

Effects of External pH Variations on Brain Presynaptic Sodium and Calcium Channels; Repercussion on the Evoked Release of Amino Acid Neurotransmitters

The effects of external pH (pH _out) variations on the Na⁺ and on the Ca²⁺ dependent fractions of the evoked amino acid neurotransmitter release were separately investigated, using GABA as a model transmitter. In [³H]GABA loaded mouse brain synaptosomes, the external acidification (pH _out6.0) markedly decreased the Na⁺ dependent fraction of [³H]GABA release evoked by veratridine (10 M) in the absence of external Ca²⁺, as well as the Ca²⁺ dependent fraction of [³H]GABA release evoked by high (20 mM) K⁺ in the absence of external Na⁺. The depolarization-induced elevation of [Na _i ] (monitored in synaptosomes loaded with the Na⁺ indicator dye, SBFI) and the depolarization-induced elevation of [Ca _i ] (monitored in synaptosomes loaded with the Ca²⁺ indicator dye fura-2) were also markedly decreased at pH _out 6. On the contrary, the external alkalinization (pH _out 8) facilitated all the above responses. A slight increase of the baseline release of the [³H]GABA was observed when pH _out was changed from 7.4 to 8. This effect was only observed in the presence of Ca²⁺. pH _out changes from 7.4 to 6 or to 7 did not modify the baseline release of the transmitter. All the effects of pH _out variations on [³H]GABA release were independent on the presence of HCO-₃. It is concluded that external H⁺ regulate amino acid neurotransmitter release by their actions on presynaptic Na⁺ channels, as well as on presynaptic Ca²⁺ channels.     

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

Rises in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca²⁺]_cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca²⁺]_cyt (Ca²⁺ buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca²⁺ or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca²⁺ signalling, we here monitor Ca²⁺ flux around the platelet by measuring net Ca²⁺ fluxes to or from the extracellular space and the intracellular Ca²⁺ stores, which act as the major sources and sinks for Ca²⁺ influx into and efflux from the cytosol, as well as monitoring the cytosolic Na⁺ concentration ([Na⁺]_cyt), which influences platelet Ca²⁺ fluxes via Na⁺/Ca²⁺ exchange. The intracellular store Ca²⁺ concentration ([Ca²⁺]_st) was monitored using Fluo-5N, the extracellular Ca²⁺ concentration ([Ca²⁺]_ext) was monitored using Fluo-4 whilst [Ca²⁺]_cyt and [Na⁺]_cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca²⁺]_cyt in the absence of extracellular Ca²⁺. PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca²⁺ release and Ca²⁺ removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca²⁺]_cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na⁺]_cyt which would be expected to reduce Ca²⁺ removal via the Na⁺/Ca²⁺ exchanger (NCX). Thrombin-evoked rises in [Na⁺]_cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn²⁺ quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca²⁺]_cyt following SERCA inhibition and either removal of extracellular Na⁺ or inhibition of Na⁺/K⁺-ATPase activity by removal of extracellular K⁺ or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca²⁺]_cyt by acceleration of SERCA activity, whilst rises in [Ca²⁺]_cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na⁺/K⁺-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na⁺]_cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca²⁺ signalling.     

Calmodulin Interacts with the Sodium/Calcium Exchanger NCX1 to Regulate Activity

Changes in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) are an important signal for various physiological activities. The Na⁺/Ca²⁺ exchangers (NCX) at the plasma membrane transport Ca²⁺ into or out of the cell according to the electrochemical gradients of Na⁺ and Ca²⁺ to modulate [Ca²⁺]_i homeostasis. Calmodulin (CaM) senses [Ca²⁺]_i changes and relays Ca²⁺ signals by binding to target proteins such as channels and transporters. However, it is not clear how calmodulin modulates NCX activity. Using CaM as a bait, we pulled down the intracellular loops subcloned from the NCX1 splice variants NCX1.1 and NCX1.3. This interaction requires both Ca²⁺ and a putative CaM-binding segment (CaMS). To determine whether CaM modulates NCX activity, we co-expressed NCX1 splice variants with CaM or CaM₁₂₃₄ (a Ca²⁺-binding deficient mutant) in HEK293T cells and measured the increase in [Ca²⁺]_i contributed by the influx of Ca²⁺ through NCX. Deleting the CaMS from NCX1.1 and NCX1.3 attenuated exchange activity and decreased membrane localization. Without the mutually exclusive exon, the exchange activity was decreased and could be partially rescued by CaM₁₂₃₄. Point-mutations at any of the 4 conserved a.a. residues in the CaMS had differential effects in NCX1.1 and NCX1.3. Mutating the first two conserved a.a. in NCX1.1 decreased exchange activity; mutating the 3^rd or 4^th conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity. Mutating the 2^nd and 3^rd conserved a.a. residues in NCX1.3 decreased exchange activity. Taken together, our results demonstrate that CaM senses changes in [Ca²⁺]_i and binds to the cytoplasmic loop of NCX1 to regulate exchange activity.     

On the special role of NCX in astrocytes: Translating Na+-transients into intracellular Ca2+ signals

As a solute carrier electrogenic transporter, the sodium/calcium exchanger (NCX1-3/SLC8A1-A3) links the trans-plasmalemmal gradients of sodium and calcium ions (Na⁺, Ca²⁺) to the membrane potential of astrocytes. Classically, NCX is considered to serve the export of Ca²⁺ at the expense of the Na⁺ gradient, defined as a “forward mode” operation. Forward mode NCX activity contributes to Ca²⁺ extrusion and thus to the recovery from intracellular Ca²⁺ signals in astrocytes. The reversal potential of the NCX, owing to its transport stoichiometry of 3 Na⁺ to 1 Ca²⁺, is, however, close to the astrocytes’ membrane potential and hence even small elevations in the astrocytic Na⁺ concentration or minor depolarisations switch it into the “reverse mode” (Ca²⁺ import/Na⁺ export). Notably, transient Na⁺ elevations in the millimolar range are induced by uptake of glutamate or GABA into astrocytes and/or by the opening of Na⁺-permeable ion channels in response to neuronal activity. Activity-related Na⁺ transients result in NCX reversal, which mediates Ca²⁺ influx from the extracellular space, thereby generating astrocyte Ca²⁺ signalling independent from InsP₃-mediated release from intracellular stores. Under pathological conditions, reverse NCX promotes cytosolic Ca²⁺ overload, while dampening Na⁺ elevations of astrocytes. This review provides an overview on our current knowledge about this fascinating transporter and its special functional role in astrocytes. We shall delineate that Na⁺-driven, reverse NCX-mediated astrocyte Ca²⁺ signals are involved neurone-glia interaction. Na⁺ transients, translated by the NCX into Ca²⁺ elevations, thereby emerge as a new signalling pathway in astrocytes.     

Key role of a PtdIns-4,5P2 micro domain in ionic regulation of the mammalian heart Na/Ca exchanger

Phosphatidylinositol biphosphate (PtdIns-4,5P₂) plays a key role in the regulation of the mammalian heart Na⁺/Ca²⁺ exchanger (NCX1) by protecting the intracellular Ca²⁺ regulatory site against H⁺_i and (H⁺_i + Na⁺_i) synergic inhibition. MgATP and MgATP-γ-S up-regulation of NCX1 takes place via the production of this phosphoinositide. In microsomes containing PtdIns-4,5P₂ incubated in the absence of MgATP and at normal [Na⁺]_i, alkalinization increases the affinity for Ca²⁺_i to the values seen in the presence of the nucleotide at normal pH; under this condition, addition of MgATP does not increase the affinity for Ca²⁺_i any further. On the other hand, prevention of Na⁺_i inhibition by alkalinization in the absence of MgATP does not take place when the microsomes are depleted of PtdIns-4,5P₂. Experiments on NCX1–PtdIns-4,5P₂ cross-coimmunoprecipitation show that the relevant PtdIns-4,5P₂ is not the overall membrane component but specifically that tightly attached to NCX1. Consequently, the highest affinity of the Ca²⁺_i regulatory site is seen in the deprotonated and PtdIns-4,5P₂-bound NCX1. Confirming these results, a PtdIns-5-kinase also cross-coimmunoprecipitates with NCX1 without losing its functional competence. These observations indicate, for the first time, the existence of a PtdIns-5-kinase in the NCX1 microdomain.     

Klotho attenuates isoproterenol-induced hypertrophic response in H9C2 cells by activating Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase and inhibiting the reverse mode of Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript>-exchanger

Cardiac hypertrophy plays a major role in heart failure and is related to patient morbidity and mortality. Calcium overloading is a main risk for cardiac hypertrophy, and Na⁺/K⁺-ATPase (NKA) has been found that it could not only regulate intracellular Na⁺ levels but also control the intracellular Ca²⁺ ([Ca²⁺]_i) level through Na⁺/Ca²⁺-exchanger (NCX). Recent studies have reported that klotho could affect [Ca²⁺]_i level. In this study, we aimed at exploring the role of klotho in improving isoproterenol-induced hypertrophic response of H9C2 cells. The H9C2 cells were randomly divided into control and isoproterenol (ISO) (10 μM) groups. Klotho protein (10 μg/ml) or NKAα2 siRNA was used to determine the changes in isoproterenol-induced hypertrophic response. The alterations of [Ca²⁺]_i level were measured by spectrofluorometry. Our results showed that H9C2 cells which were treated with isoproterenol presented a higher level of [Ca²⁺]_i and hypertrophic gene expression at 24 and 48 h compared with the control group. Moreover, the expressions of NKAα1 and NKAα2 were both increased in control and ISO groups after treating with klotho protein; meanwhile, the NKA activity was increased and NCX activity was decreased after treatment. Consistently, the [Ca²⁺]_i level and hypertrophic gene expression were decreased in ISO group after klotho protein treatment. However, these effects were both prevented by transfecting with NKAα2 siRNA. In conclusion, these findings demonstrated that klotho inhibits isoproterenol-induced hypertrophic response in H9C2 cells by activating NKA and inhibiting the reverse mode of NCX and this effect may be associated with the upregulation of NKAα2 expression.     

Sodium-Dependent Efflux of [3H]GABA from Synaptosomes Probably Related to Mitochondrial Calcium Mobilization

It has been suggested that mitochondria might modify transmitter release through the control of intracellular Ca²⁺levels. Treatments known to inhibit Ca²⁺retention by mitochondria lead to an increased transmitter liberation in the absence of external Ca²⁺, both at the frog neuromuscular junction and from isolated nerve endings. Sodium ions stimulate Ca²⁺efflux from mitochondria isolated from excitable tissues. In the present study, the effect of increasing internal Na⁺ levels on [³H]γ-aminobutyric acid ([³H]GABa) release from isolated nerve endings is reported. Results show that the efflux of [³H]GABA from prelabeled synaptosomes is stimulated by ouabain, veratrine, gramicidin D, and K⁺-free medium, which increase the internal sodium concentration. This effect was not observed when Na⁺ was omitted from the incubation medium and it was independent of external Ca²⁺, the experiments having been performed in a Ca²⁺-free, EGTA-containing medium. Since preincubation of synaptosomes with 2,4-diaminobutyric acid did not prevent the stimulatory effect of increased internal Na⁺ levels on [³H]GABA efflux, it appears to be unrelated to an enhanced activity of the outward carrier-mediated GABA transport. These results suggest that the augmented release of [³H]GABA may be due to an increased Ca²⁺efflux from mitochondria eiicited by the accumulation of Na⁺ at the nerve endings. Sandoval M. E. Sodium-dependent efflux of [³H]GABA from synaptosomes probably related to mitochondrial calcium mobilization. J. Neurochem. 35 , 915–921 (1980).     

A role for calcium/calmodulin kinase(s) in the regulation of GABA exocytosis

A possible role for protein kinases in the regulation of GABA exocytosis in nerve endings was investigated. The effect on the release of the radioactive neurotransmitter ([³H]GABA) from mouse brain synaptosomes of several protein kinase inhibitors was estimated after treatment with 37 mM K⁺ in the absence of external Na⁺, a condition under which [³H]GABA release is completely Ca²⁺ dependent. Among the inhibitors one group inhibit the kinases by binding to the catalytic site (i.e. staurosporine and H7) and others (TFP, sphingosine and W7) act on the regulatory site of protein kinases. The compounds of the second group, which are reported to inhibit calmodulin dependent events and the increase in cytosolic Ca²⁺ (Ca _i ) induced by high K⁺ depolarization, were the most efficient inhibitors of [³H]GABA release. The selective inhibitor of CaMPK II, KN-62, also markedly diminished [³H]GABA release as well as the increase in Ca _i induced by high K⁺. The kinase inhibitors from the first group that are unable to diminish the increase in Ca _i induced by high K⁺ were also less efficient inhibitors of [³H]GABA release even at high concentrations. The present results indicate that at the doses tested all the drugs inhibit to some extent the release of the Ca²⁺ dependent fraction of [³H]GABA perhaps by inhibiting a CaMPK II mediated phosphorylation step triggered by depolarization and facilitated by the elevation of Ca _i . In addition, the second group of antagonists and KN-62 inhibit the elevation of Ca _i to high K⁺ thus exhibiting a higher efficiency on [³H]GABA release than the first group of antagonists.     

NCX activity generates spontaneous Ca2+ oscillations in the astrocytic leaflet microdomain

The synergy between synaptic Glu release and astrocytic Glu-Na⁺ symport is essential to the signalling function of the tripartite synapse. Here we used kinetic data of astrocytic Glu transporters (EAAT) and the Na⁺/Ca²⁺ exchanger (NCX) to simulate Glu release, Glu uptake and subsequent Na⁺ and Ca²⁺ dynamics in the astrocytic leaflet microdomain following single release event. Model simulations show that Glu-Na⁺ symport differently affect intracellular [Na⁺] in synapses with different extent of astrocytic coverage. Surprisingly, NCX activity alone has been shown to generate markedly stable, spontaneous Ca²⁺ oscillation in the astrocytic leaflet. These on-going oscillations appear when NCX operates either in the forward or reverse direction. We conjecture that intrinsic NCX activity may play a prominent role in the generation of astrocytic Ca²⁺ oscillations.     

A Transmural Gradient in the Cardiac Na/K Pump Generates a Transmural Gradient in Na/Ca Exchange

We previously demonstrated a transmural gradient in Na/K pump current (I _P) and [Na⁺] _i , with the highest maximum I _P and lowest [Na⁺] _i in epicardium. The present study examines the relationship between the transmural gradient in I _P and Na/Ca exchange (NCX). Myocytes were isolated from canine left ventricle. Whole-cell patch clamp was used to measure current generated by NCX (I _NCX) and inward background calcium current (I _ibCa), defined as inward current through Ca²⁺ channels less outward current through Ca²⁺-ATPase. When resting myocytes from endocardium (Endo), midmyocardium (Mid) or epicardium (Epi) were studied in the same conditions, I _NCX was the same and I _ibCa was zero. Moreover, Western blots were consistent with NCX protein being uniform across the wall. However, the gradient in [Na⁺] _i , with I _ibCa = 0, should create a gradient in [Ca²⁺] _i . To test this hypothesis, we measured resting [Ca²⁺] _i using two methods, based on either transport or the Ca²⁺-sensitive dye Fura2. Both methods demonstrated a significant transmural gradient in resting [Ca²⁺] _i , with Endo > Mid > Epi. This gradient was eliminated by exposing Epi to sufficient ouabain to partially inhibit Na/K pumps, thus increasing [Na⁺] _i to values similar to those in Endo. These data support the existence of a transmural gradient for Ca²⁺ removal by NCX. This gradient is not due to differences in expression of NCX; rather, it is generated by a transmural gradient in [Na⁺] _i , which is due to a transmural gradient in plasma membrane expression of the Na/K pump.     

Ionic Requirements for the Specific Binding of [3H]GBR 12783 to a Site Associated with the Dopamine Uptake Carrier

At 0°C, when Na⁺ was the only cation present in the incubation medium, increasing the Na⁺ concentration from 3 to 10 mM enhanced the affinity of [³H]l-[2-(di-phenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([³H]GBR 12783) for the specific binding site present in rat striatal membranes without affecting the 5_max. For higher Na⁺ concentrations, specific binding values plateaued and then slightly decreased at 130 mM Na⁺. In a 10 mM Na⁺ medium, the K_D and the B_max were, respectively, 0.23 nM and 12.9 pmol/mg of protein. In the presence of 0.4 nM [³H]GBR 12783, the half-maximal specific binding occurred at 5 mM Na⁺. A similar Na⁺ dependence was observed at 20°C. Scatchard plots indicated that K⁺, Ca²⁺, Mg²⁺, and Tris⁺ acted like competitive inhibitors of the specific binding of [³H]GBR 12783. The inhibitory potency of various cations (K⁺, Ca²⁺, Mg²⁺, Tris⁺, Li⁺ and choline) was enhanced when the Na⁺ concentration was decreased from 130 to 10 mM. In a 10 mM Na⁺ medium, the rank order of inhibitory potency was Ca²⁺ (0.13 mM) > Mg²⁺ > Tris⁺ > K⁺ (15 mM). The requirement for Na⁺ was rather specific, because none of the other cations acted as a substitute for Na⁺. No anionic requirement was found: Cl^-, Br^-, and F^- were equipotent. These results suggest that low Na⁺ concentrations are required for maximal binding; higher Na⁺ concentrations protect the specific binding site against the inhibitory effect of other cations.     

Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

Involvement of the Na+/Ca2+ exchanger isoform 1 (NCX1) in Neuronal Growth Factor (NGF)-induced Neuronal Differentiation through Ca2+-dependent Akt Phosphorylation

NGF induces neuronal differentiation by modulating [Ca²⁺]_i. However, the role of the three isoforms of the main Ca²⁺-extruding system, the Na⁺/Ca²⁺ exchanger (NCX), in NGF-induced differentiation remains unexplored. We investigated whether NCX1, NCX2, and NCX3 isoforms could play a relevant role in neuronal differentiation through the modulation of [Ca²⁺]_i and the Akt pathway. NGF caused progressive neurite elongation; a significant increase of the well known marker of growth cones, GAP-43; and an enhancement of endoplasmic reticulum (ER) Ca²⁺ content and of Akt phosphorylation through an early activation of ERK1/2. Interestingly, during NGF-induced differentiation, the NCX1 protein level increased, NCX3 decreased, and NCX2 remained unaffected. At the same time, NCX total activity increased. Moreover, NCX1 colocalized and coimmunoprecipitated with GAP-43, and NCX1 silencing prevented NGF-induced effects on GAP-43 expression, Akt phosphorylation, and neurite outgrowth. On the other hand, the overexpression of its neuronal splicing isoform, NCX1.4, even in the absence of NGF, induced an increase in Akt phosphorylation and GAP-43 protein expression. Interestingly, tetrodotoxin-sensitive Na⁺ currents and 1,3-benzenedicarboxylic acid, 4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester-detected [Na⁺]_i significantly increased in cells overexpressing NCX1.4 as well as ER Ca²⁺ content. This latter effect was prevented by tetrodotoxin. Furthermore, either the [Ca²⁺]_i chelator(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (BAPTA-AM) or the PI3K inhibitor LY 294002 prevented Akt phosphorylation and GAP-43 protein expression rise in NCX1.4 overexpressing cells. Moreover, in primary cortical neurons, NCX1 silencing prevented Akt phosphorylation, GAP-43 and MAP2 overexpression, and neurite elongation. Collectively, these data show that NCX1 participates in neuronal differentiation through the modulation of ER Ca²⁺ content and PI3K signaling.     

Evidence for a similar compartmentation of recaptured and endogenously synthesized dopamine in striatal synaptosomes

The aim of the present study was to compare the release pattern of [³H]dopamine ([³H]DA) originated from [³H]tyrosine or by uptake in striatal synaptosomes. Synaptosomes prelabeled either with [³H]DA or with [³H]tyrosine were superfused in three conditions stimulating DA release by different mechanisms: (1) depolarization with high K⁺; (2) inversion of the Na⁺ gradient across the plasma membrane; (3) exposure tod-amphetamine. Since DA contained in different pools may exit from nerve endings by different processes, DA release was analyzed in the presence or in the absence of nomifensine which allows discrimination between carrier-mediated and carrier-independent processes. The pattern of DA release in the three conditions tested was identical, whether [³H]DA originated from synthesis or from uptake. Nomifensine did not affect the high-K⁺-induced release and inhibited that induced by the other two stimuli. The results suggest that newly synthesized and recaptured DA have a similar compartmentation in nerve endings.     

Ionic regulation of the cardiac sodium-calcium exchanger

The Na⁺-Ca²⁺ exchanger (NCX) links transmembrane movements of Ca²⁺ ions to the reciprocal movement of Na+ ions. It normally functions primarily as a Ca²⁺ efflux mechanism in excitable tissues such as the heart, but it can also mediate Ca²⁺ influx under certain conditions. Na⁺ and Ca²⁺ ions exert complex regulatory effects on NCX activity. Ca²⁺ binds to two regulatory sites in the exchanger's central hydrophilic domain, and this interaction is normally essential for activation of exchange activity. High cytosolic Na⁺ concentrations, however, can induce a constitutive activity that by-passes the need for allosteric Ca²⁺ activation. Constitutive NCX activity can also be induced by high levels of phopshotidylinositol-4,5-bisphosphate (PIP₂) and by mutations affecting the regulatory calcium binding domains. In addition to promoting constitutive activity, high cytosolic Na⁺ concentrations also induce an inactivated state of the exchanger (Na⁺-dependent inactivation) that becomes dominant when cytosolic pH and PIP₂ levels fall. Na+-dependent inactivation may provide a means of protecting cells from Ca²⁺ overload due to NCX-mediated Ca²⁺ influx during ischemia.