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.     

Regulation of the Na+/Ca2+ Exchanger by Pyridine Nucleotide Redox Potential in Ventricular Myocytes

The cardiac Na⁺/Ca²⁺ exchanger (NCX) is the major Ca²⁺ efflux pathway on the sarcolemma, counterbalancing Ca²⁺ influx via L-type Ca²⁺ current during excitation-contraction coupling. Altered NCX activity modulates the sarcoplastic reticulum Ca²⁺ load and can contribute to abnormal Ca²⁺ handling and arrhythmias. NADH/NAD⁺ is the main redox couple controlling mitochondrial energy production, glycolysis, and other redox reactions. Here, we tested whether cytosolic NADH/NAD⁺ redox potential regulates NCX activity in adult cardiomyocytes. NCX current (I_NCX), measured with whole cell patch clamp, was inhibited in response to cytosolic NADH loaded directly via pipette or increased by extracellular lactate perfusion, whereas an increase of mitochondrial NADH had no effect. Reactive oxygen species (ROS) accumulation was enhanced by increasing cytosolic NADH, and NADH-induced I_NCX inhibition was abolished by the H₂O₂ scavenger catalase. NADH-induced ROS accumulation was independent of mitochondrial respiration (rotenone-insensitive) but was inhibited by the flavoenzyme blocker diphenylene iodonium. NADPH oxidase was ruled out as the effector because I_NCX was insensitive to cytosolic NADPH, and NADH-induced ROS and I_NCX inhibition were not abrogated by the specific NADPH oxidase inhibitor gp91ds-tat. This study reveals a novel mechanism of NCX regulation by cytosolic NADH/NAD⁺ redox potential through a ROS-generating NADH-driven flavoprotein oxidase. The mechanism is likely to play a key role in Ca²⁺ homeostasis and the response to alterations in the cytosolic pyridine nucleotide redox state during ischemia-reperfusion or other cardiovascular diseases.     

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.     

Mechanism of the persistent sodium current activator veratridine-evoked Ca2+ elevation: implication for epilepsy

Although the role of Na⁺ in several aspects of Ca²⁺ regulation has already been shown, the exact mechanism of intracellular Ca²⁺ concentration ([Ca²⁺]_i) increase resulting from an enhancement in the persistent, non‐inactivating Na⁺ current (I_Na,P), a decisive factor in certain forms of epilepsy, has yet to be resolved. Persistent Na⁺ current, evoked by veratridine, induced bursts of action potentials and sustained membrane depolarization with monophasic intracellular Na⁺ concentration ([Na⁺]_i) and biphasic [Ca²⁺]_i increase in CA1 pyramidal cells in acute hippocampal slices. The Ca²⁺ response was tetrodotoxin‐ and extracellular Ca²⁺‐dependent and ionotropic glutamate receptor‐independent. The first phase of [Ca²⁺]_i rise was the net result of Ca²⁺ influx through voltage‐gated Ca²⁺ channels and mitochondrial Ca²⁺ sequestration. The robust second phase in addition involved reverse operation of the Na⁺–Ca²⁺ exchanger and mitochondrial Ca²⁺ release. We excluded contribution of the endoplasmic reticulum. These results demonstrate a complex interaction between persistent, non‐inactivating Na⁺ current and [Ca²⁺]_i regulation in CA1 pyramidal cells. The described cellular mechanisms are most likely part of the pathomechanism of certain forms of epilepsy that are associated with I_Na,P. Describing the magnitude, temporal pattern and sources of Ca²⁺ increase induced by I_Na,P may provide novel targets for antiepileptic drug therapy.     

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).     

Calcium signalling in Drosophila photoreceptors measured with GCaMP6f

Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca²⁺ influx in response to light, but whether Ca²⁺ is released from internal stores remains controversial. We generated flies expressing GCaMP6f in their photoreceptors and measured Ca²⁺ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10–25 ms, reached 50% F_max with ∼1200 effectively absorbed photons and saturated (ΔF/F₀ ∼ 10–20) with 10000–30000 photons. In Ca²⁺ free bath, smaller (ΔF/F₀ ∼4), long latency (∼200 ms) light-induced Ca²⁺ rises were still detectable. These were unaffected in InsP₃ receptor mutants, but virtually eliminated when Na⁺ was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca²⁺ free rises were also eliminated in Na⁺/Ca²⁺ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca²⁺ free rises are strictly dependent on Na⁺ influx and activity of the exchanger, suggesting they reflect re-equilibration of Na⁺/Ca²⁺ exchange across plasma or intracellular membranes following massive Na⁺ influx. Any tiny Ca²⁺ free rise remaining without exchanger activity was equivalent to <10 nM (ΔF/F₀ ∼0.1), and unlikely to play any role in phototransduction.     

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⁺.     

Tachyphylaxis to the inhibitory effect of L-type channel blockers on ACh-induced [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> oscillations in porcine tracheal myocytes

Summary Discrepancies about the role of L-type voltage-gated calcium channels (VGCC) in acetylcholine (ACh)-induced [Ca²⁺]_i oscillations in tracheal smooth muscle cells (TSMCs) have been seen in recent reports. We demonstrate here that ACh-induced [Ca²⁺]_i oscillations in TMCS were reversibly inhibited by three VGCC blockers, nicardipine, nifedipine and verapamil. Prolonged (several minutes) application of VGCC blockers, led to tachyphylaxis; that is, [Ca²⁺]_i oscillations resumed, but at a lower frequency. Brief (15–30 s) removal of VGCC blockers re-sensitized [Ca²⁺]_i oscillations to inhibition by the agents. Calcium oscillations tolerant to VGCC blockers were abolished by KB-R7943, an inhibitor of the reverse mode of Na⁺/Ca²⁺ exchanger (NCX). KB-R7943 alone also abolished ACh-induced [Ca²⁺]_i oscillations. Enhancement of the reverse mode of NCX via removing extracellular Na⁺ reversed inhibition of ACh-induced [Ca²⁺]_i oscillations by VGCC blockers. Inhibition of non-selective cation channels using Gd³⁺ slightly reduced the frequency of ACh-induced [Ca²⁺]_i oscillations, but did not prevent the occurrence of tachyphylaxis. Altogether, these results suggest that VGCC and the reverse mode of NCX are two primary Ca²⁺ entry pathways for maintaining ACh-induced [Ca²⁺]_i oscillations in TSMCs. The two pathways complement each other, and may account for tachyphylaxis of ACh-induced [Ca²⁺]_i oscillations to VGCC blockers.     

The specific Na(+)/Ca(2+) exchange inhibitor SEA0400 prevents nitric oxide-induced cytotoxicity in SH-SY5Y cells

The Na⁺/Ca²⁺ exchanger (NCX) plays a role in the regulation of intracellular Ca²⁺ levels, and nitric oxide (NO) is involved in many pathological conditions including neurodegenerative disorders. We have previously found that sodium nitroprusside (SNP), an NO donor, causes apoptotic-like cell death in cultured glial cells via NCX-mediated pathways and the mechanism for NO-induced cytotoxicity is cell type-dependent. The present study examined using the specific NCX inhibitor 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400) whether NCX is involved in NO-induced injury in cultured neuronal cells. The treatment of neuroblastoma SH-SY5Y cells with SNP resulted in apoptosis and the cytotoxicity was blocked by the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase inhibitor U0126 and the p38 MAP kinase (MAPK) inhibitor SB203580, but not by the c-Jun N-terminal kinase (JNK) inhibitor SP60012. SNP increased Ca²⁺ influx and intracellular Ca²⁺ levels. In addition, SNP increased ERK and p38 MAPK phosphorylation, and production of reactive oxygen species (ROS) in an extracellular Ca²⁺-dependent manner. These effects of SNP were prevented by SEA0400. SNP-induced cytotoxicity was not affected by inhibitors of the Ca²⁺, Na⁺ and store-operated/capacitative channels. Moreover, SNP-induced increase in intracellular Ca²⁺ levels, ROS production and decrease in cell viability were blocked by a cGMP-dependent protein kinase (PKG) inhibitor. These results suggest that Ca²⁺ influx via the reverse of NCX is involved in the cascade of NO-induced neuronal apoptosis and NO activates the NCX through guanylate cyclase/PKG pathway.     

Roles of transient receptor potential canonical (TRPC) channels and reverse-mode Na/Ca exchanger on cell proliferation in human cardiac fibroblasts: Effects of transforming growth factor β1

Expression of transient receptor potential canonical channels (TRPC) and the effects of transforming growth factor-β1 (TGF-β1) on Ca²⁺ signals and fibroblast proliferation were investigated in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, western blot, immunocytochemical analysis, and intracellular Ca²⁺ concentration [Ca²⁺]_i measurement were applied. Cell proliferation and cell cycle progression were assessed using MTT assays and fluorescence activated cell sorting. Human cardiac fibroblasts have the expression of TRPC1,3,4,6 mRNA and proteins. 1-oleoyl-2-acetyl-sn-glycerol (OAG) and thapsigargin induced extracellular Ca²⁺-mediated [Ca²⁺]_i rise. siRNA for knock down of TRPC6 reduced OAG-induced Ca²⁺ entry. Hyperforin as well as angiotensin II (Ang II) induced Ca²⁺ entry. KB-R7943, a reverse-mode Na⁺/Ca²⁺ exchanger (NCX) inhibitor, and/or replacement of Na⁺ with NMDG⁺ inhibited thapsigargin-, OAG- and Ang II-induced Ca²⁺ entry. Treatment with TGF-β1 increased thapsigargin-, OAG- and Ang II-induced Ca²⁺ entry with an enhancement of TRPC1,6 protein expression, suppressed by KB-R7943. TGF-β1 and AngII promoted cell cycle progression from G0/G1 to S/G2/M and cell proliferation. A decrease of the extracellular Ca²⁺ and KB-R7943 suppressed it. Human cardiac fibroblasts contain several TRPC-mediated Ca²⁺ influx pathways, which activate the reverse-mode NCX. TGF-β1 enhances the Ca²⁺ influx pathways requiring Ca²⁺ signals for its effect on fibroblast proliferation.     

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.     

Mitochondrial Ca influx and efflux rates in guinea pig cardiac mitochondria:Low and high affinity effects of cyclosporine A

Ca²⁺ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca²⁺ is controlled primarily by the mitochondrial Ca²⁺ uniporter and the mitochondrial Na⁺/Ca²⁺ exchanger, influencing NADH production through Ca²⁺-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca²⁺-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca²⁺ release. Here we selectively measure Ca²⁺ influx rate through the mitochondrial Ca²⁺ uniporter and Ca²⁺ efflux rates through Na⁺-dependent and Na⁺-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na⁺/Ca²⁺ exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ_m loss, Ca²⁺ release, NADH oxidation, swelling) of high extramitochondrial Ca²⁺ additions, allowing mitochondria to tolerate total mitochondrial Ca²⁺ loads of > 400 nmol/mg protein. For Ca²⁺ pulses up to 15 μM, Na⁺-independent Ca²⁺ efflux through the permeability transition pore accounted for ~ 5% of the total Ca²⁺ efflux rate compared to that mediated by the mitochondrial Na⁺/Ca²⁺ exchanger (in 5 mM Na⁺). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na⁺/Ca²⁺ exchanger-mediated Ca²⁺ efflux at higher concentrations (IC₅₀ = 2 μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~ 40% at 10 μM cyclosporine A, while having no effect on the mitochondrial Ca²⁺ uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca²⁺ load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Modulation of the cardiac Na+-Ca2+ exchanger by cytoplasmic protons: Molecular mechanisms and physiological implications

A precise temporal and spatial control of intracellular Ca²⁺ concentration is essential for a coordinated contraction of the heart. Following contraction, cardiac cells need to rapidly remove intracellular Ca²⁺ to allow for relaxation. This task is performed by two transporters: the plasma membrane Na⁺-Ca²⁺ exchanger (NCX) and the sarcoplasmic reticulum (SR) Ca²⁺‐ATPase (SERCA). NCX extrudes Ca²⁺ from the cell, balancing the Ca²⁺entering the cytoplasm during systole through L-type Ca²⁺ channels. In parallel, following SR Ca²⁺ release, SERCA activity replenishes the SR, reuptaking Ca²⁺ from the cytoplasm.The activity of the mammalian exchanger is fine-tuned by numerous ionic allosteric regulatory mechanisms. Micromolar concentrations of cytoplasmic Ca²⁺ potentiate NCX activity, while an increase in intracellular Na⁺ levels inhibits NCX via a mechanism known as Na⁺-dependent inactivation. Protons are also powerful inhibitors of NCX activity. By regulating NCX activity, Ca²⁺, Na⁺ and H⁺ couple cell metabolism to Ca²⁺ homeostasis and therefore cardiac contractility. This review summarizes the recent progress towards the understanding of the molecular mechanisms underlying the ionic regulation of the cardiac NCX with special emphasis on pH modulation and its physiological impact on the heart.     

Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger

Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na⁺/Ca²⁺ exchanger (NCX) operating in the Ca²⁺-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca²⁺ influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na⁺-K⁺-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans'' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving unregulated thrombin signaling.     

Sodium-calcium exchange in renal epithelial cells: Dependence on cell sodium and competitive inhibition by magnesium

Summary Kinetic properties of Na⁺–Ca²⁺ exchange in a renal epithelial cell line (LLC-MK₂) were assessed by measuring cytosolic free Ca²⁺ with fura-2 and⁴⁵Ca²⁺ influx. Replacing external Na⁺ with K⁺ produced relatively small increases in free Ca²⁺ and⁴⁵Ca²⁺ uptake unless the cells were incubated with ouabain. Ouabain markedly increased cell Na⁺ and strongly potentiated the effect of replacing external Na⁺ with K⁺ on free Ca²⁺ and⁴⁵Ca²⁺ uptake.⁴⁵Ca²⁺ influx in 140mm K⁺ or N-methyl-d-glucamine minus influx in 140mm Na⁺ was used to quantify Na⁺–Ca²⁺ exchange activity of Na⁺-loaded cells. The dependence of exchange on cell Na⁺ was sigmoidal; theK _0.5 was 26±3 mmol/liter cell water space, and the Hill coefficient was 3.1±0.2. The kinetic features of the dependence of exchange on cell Na⁺ partly account for the small increase in Ca²⁺ influx when all external Na⁺ is replaced by K⁺. Besides raising cell Na⁺ ouabain appears to activate the exchanger. Magnesium competitively inhibited exchange activity. The potency of Mg²⁺ was 8.2-fold lower with potassium instead of N-methyl-d-glucamine or choline as the replacement for external Na⁺. Potassium also increased theV _max of exchange by 86% and had no effect on theK _m for Ca²⁺. The exchanger does not cause detectable²²Na⁺–Mg²⁺ exchange and does not appear to require K⁺ or transport⁸⁶Rb⁺. Although exchange activity was plentiful in the epithelial cells from monkey kidney, others from amphibian, canine, opossum, and porcine kidney had no detectable exchange activity. All of the measured kinetic properties of Na⁺–Ca²⁺ exchange in the renal epithelial cells are very similar to those of the exchanger in rat aortic myocytes.     

Mitochondrial localization of NCXs: Balancing calcium and energy homeostasis

It is well established that mitochondria are the main source of ATP production within cells. However, mitochondria have other remarkable functions, serving as important modulators of cellular Ca²⁺ signaling, and it is now generally recognized that control over Ca²⁺ homeostasis is intrinsically interwoven with mitochondrial abilities to adjust and tune ATP production. In this review, we describe the mechanisms that mitochondria use to balance Ca²⁺ homeostasis maintenance and cell energy metabolism. In recent years, the knowledge on the molecular machinery mediating Ca²⁺ influx/efflux has been improved and, albeit still open to further investigations, several lines of evidence converge on the hypothesis that plasma membrane Na⁺/Ca²⁺ exchanger (NCX) isoforms are also expressed at the mitochondrial level, where they contribute to the Ca²⁺ and Na⁺ homeostasis maintenance. In particular, the connection between mitochondrial NCX activity and metabolic substrates utilization is further discussed here. We also briefly focus on the alterations of both mitochondrial Ca²⁺ handling and cellular bioenergetics in neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease.     

Suppression of programmed neuronal death by a thapsigargin-induced Ca2+ influx

Rat sympathetic neurons undergo programmed cell death (PCD) in vitro and in vivo when they are deprived of nerve growth factor (NGF). Chronic depolarization of these neurons in cell culture with elevated concentrations of extracellular potassium ([K⁺]_o) prevents this death. The effect of prolonged depolarization on neuronal survival is thought to be mediated by a rise of intracellular calcium concentration ([Ca²⁺]_i) caused by Ca²⁺ influx through voltage-gated channels. In this report we investigate the effects of chronic treatment of rat sympathetic neurons with thapsigargin, an inhibitor of intracellular Ca²⁺ sequestration. In medium containing a normal concentration of extracellular Ca²⁺ ([Ca²⁺]_o), thapsigargin caused a sustained rise of intracellular Ca²⁺ concentration and partially blocked death of NGF-deprived cells. Elevating [Ca²⁺]_o in the presence of thapsigargin further increased [Ca²⁺]_i, suggesting that the sustained rise of [Ca²⁺]_i was caused by a thapsigargin-induced Ca²⁺ influx. This treatment potentiated the effect of thapsigargin on survival. The dihydropyridine Ca²⁺ channel antagonist, nifedipine, blocked both a sustained elevation of [Ca²⁺]_i and enhanced survival caused by depolarization with elevated [K⁺]_o, suggesting that these effects are mediated by Ca²⁺ influx through L-type channels. Nifedipine did not block the sustained rise of [Ca²⁺]_i or enhanced survival caused by thapsigargin treatment, indicating that these effects were not mediated by influx of Ca²⁺ through L-type channels. These results provide additional evidence that increased [Ca²⁺]_i can suppress neuronal PCD and identify a novel method for chronically raising neuronal [Ca²⁺]_i for investigation of this and other Ca²⁺-dependent phenomena. © 1995 John Wiley & Sons, Inc.     

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.     

Persistent Na+ influx drives L-type channel resting Ca2+ entry in rat melanotrophs

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) remain unknown. We analyzed mechanisms regulating resting [Ca²⁺]_i in dissociated rat melanotrophs by Ca²⁺-imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca²⁺ channels (VGCCs) as well as removal of extracellular Ca²⁺ resulted in a rapid and reversible decrease in [Ca²⁺]_i, indicating constitutive Ca²⁺ influx through L-type VGCCs. Reduction of extracellular Na⁺ concentration (replacement with NMDG⁺) similarly decreased resting [Ca²⁺]_i. When cells were champed at –80 mV, decrease in the extracellular Na⁺ resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na⁺ caused hyperpolarisation. The holding current shifted in negative direction when extracellular K⁺ concentration was increased from 5 mM to 50 mM in the presence of K⁺ channel blockers, Ba²⁺ and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca²⁺]_i. These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca²⁺]_i in rat melanotrophs.