Ncx3 gene ablation impairs oligodendrocyte precursor response and increases susceptibility to experimental autoimmune encephalomyelitis.

The Na(+) /Ca(2+) exchanger NCX3, recently identified as a myelin membrane component, is involved in the regulation of [Ca(2+) ]i during oligodendrocyte maturation. Here NCX3 involvement was studied in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Western blotting and quantitative colocalization studies performed in wild-type ncx3(+/+) mice at different stages of EAE disease showed that NCX3 protein was intensely upregulated during the chronic stage, where it was intensely coexpressed with the oligodendrocyte precursor cells (OPC) marker NG2 and the premyelinating marker CNPase. Moreover, MOG35-55 -immunized mice lacking the ncx3 gene displayed not only a reduced diameter of axons and an intact myelin ring number but also a dramatic decrease in OPC and pre-myelinating cells in the white matter of the spinal cord when compared with ncx3(+/+) . Accordingly, ncx3(-/-) and ncx3(+/-) mutants developed early onset of EAE and more severe clinical symptoms. Interestingly, cytofluorimetric analysis revealed that during the peak stage of the disease, the number of immune T-cell subsets in ncx3(-/-) mice, was not statistically different from that measured in ncx3(+/+) . Our findings demonstrate that knocking-out NCX3 impairs oligodendrocyte response and worsens clinical symptoms in EAE without altering the immune T-cell population. GLIA 2016;64:1124-1137.

Heterozygous inactivation of plasma membrane Ca(2+)-ATPase in mice increases glucose-induced insulin release and beta cell proliferation, mass and viability.

AIMS/HYPOTHESIS: Calcium plays an important role in the process of glucose-induced insulin release in pancreatic beta cells. These cells are equipped with a double system responsible for Ca(2+) extrusion--the Na/Ca exchanger (NCX) and the plasma membrane Ca(2+)-ATPase (PMCA). We have shown that heterozygous inactivation of NCX1 in mice increased glucose-induced insulin release and stimulated beta cell proliferation and mass. In the present study, we examined the effects of heterozygous inactivation of the PMCA on beta cell function. METHODS: Biological and morphological methods (Ca(2+) imaging, Ca(2+) uptake, glucose metabolism, insulin release and immunohistochemistry) were used to assess beta cell function and proliferation in Pmca2 (also known as Atp2b2) heterozygous mice and control littermates ex vivo. Blood glucose and insulin levels were also measured to assess glucose metabolism in vivo. RESULTS: Pmca (isoform 2) heterozygous inactivation increased intracellular Ca(2+) stores and glucose-induced insulin release. Moreover, increased beta cell proliferation, mass, viability and islet size were observed in Pmca2 heterozygous mice. However, no differences in beta cell glucose metabolism, proinsulin immunostaining and insulin content were observed. CONCLUSIONS/INTERPRETATION: The present data indicates that inhibition of Ca(2+) extrusion from the beta cell and its subsequent intracellular accumulation stimulates beta cell function, proliferation and mass. This is in agreement with our previous results observed in mice displaying heterozygous inactivation of NCX, and indicates that inhibition of Ca(2+) extrusion mechanisms by small molecules in beta cells may represent a new approach in the treatment of type 1 and type 2 diabetes.

Intracellular dyssynchrony of diastolic cytosolic [Ca²âº] decay in ventricular cardiomyocytes in cardiac remodeling and human heart failure.

RATIONALE: Synchronized release of Ca²âº into the cytosol during each cardiac cycle determines cardiomyocyte contraction. OBJECTIVE: We investigated synchrony of cytosolic [Ca²âº] decay during diastole and the impact of cardiac remodeling. METHODS AND RESULTS: Local cytosolic [Ca²âº] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca²âº] decay based on the local time constants of decay (TAUlocal). The SD of TAUlocal as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca²âº release. Stimulation of sarcoplasmic reticulum Ca²âº ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAUlocal significantly more in slowCaR, thus altering the relationship between SD of TAUlocal and global [Ca²âº] decay (TAUglobal). Na⁺/Ca²âº exchanger inhibitor SEA0400 prolonged TAUlocal similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca²âº uniporter blocker Ru360. Variation in TAUlocal was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAUlocal correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAUlocal was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. CONCLUSIONS: In cardiomyocytes, cytosolic [Ca²âº] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca²âº] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.

The Na+/Ca2+ exchanger NCX3 mediates Ca2+ entry into matrix vesicles to facilitate initial steps of mineralization in osteoblasts.

Matrix vesicles (MVs) provide the initial site for amorphous hydroxyapatite (HA) formation within mineralizing osteoblasts. Although Na+/Ca2+ exchanger isoform-3 (NCX3, SLC8A3) was presumed to function as major Ca2+ transporter responsible for Ca2+ extrusion out of osteoblast into the calcifying bone matrix, its presence and functional role in MVs have not been investigated. In this study, we investigated the involvement of NCX3 in MV-mediated mineralization process and its impact on bone formation. Using differentiated MC3T3-E1 cells, we demonstrated that NCX3 knockout in these cells resulted in a significant reduction of Ca2+ deposition due to reduced Ca2+ entry within the MVs, leading to impaired mineralization. Consequently, the capacity of MVs to promote extracellular HA formation was diminished. Moreover, primary osteoblast isolated from NCX3 deficient mice (NCX3-/-) exhibits reduced mineralization efficacy without any effect on osteoclast activity. To validate this in vitro finding, µCT analysis revealed a substantial decrease in trabecular bone mineral density in both genders of NCX3-/- mice, thus supporting the critical role of NCX3 in facilitating Ca2+ uptake into the MVs to initiate osteoblast-mediated mineralization. NCX3 expression was also found to be the target of downregulation by inflammatory mediators in vitro and in vivo. This newfound understanding of NCX3's functional role in MVs opens new avenues for therapeutic interventions aimed at enhancing bone mineralization and treating mineralization-related disorders.

A new concept: Aß1-42 generates a hyperfunctional proteolytic NCX3 fragment that delays caspase-12 activation and neuronal death.

Although the amyloid-ß(1-42) (Aß(1-42)) peptide involved in Alzheimer's disease is known to cause a dysregulation of intracellular Ca(2+) homeostasis, its molecular mechanisms still remain unclear. We report that the extracellular-dependent early increase (30 min) in intracellular calcium concentration ([Ca(2+)](i)), following Aß(1-42) exposure, caused the activation of calpain that in turn elicited a cleavage of the Na(+)/Ca(2+) exchanger isoform NCX3. This cleavage generated a hyperfunctional form of the antiporter and increased NCX currents (I(NCX)) in the reverse mode of operation. Interestingly, this NCX3 calpain-dependent cleavage was essential for the Aß(1-42)-dependent I(NCX) increase. Indeed, the calpain inhibitor calpeptin and the removal of the calpain-cleavage recognition sequence, via site-directed mutagenesis, abolished this effect. Moreover, the enhanced NCX3 activity was paralleled by an increased Ca(2+) content in the endoplasmic reticulum (ER) stores. Remarkably, the silencing in PC-12 cells or the knocking-out in mice of the ncx3 gene prevented the enhancement of both I(NCX) and Ca(2+) content in ER stores, suggesting that NCX3 was involved in the increase of ER Ca(2+) content stimulated by Aß(1-42). By contrast, in the late phase (72 h), when the NCX3 proteolytic cleavage abruptly ceased, the occurrence of a parallel reduction in ER Ca(2+) content triggered ER stress, as revealed by caspase-12 activation. Concomitantly, the late increase in [Ca(2+)](i) coincided with neuronal death. Interestingly, NCX3 silencing caused an earlier activation of Aß(1-42)-induced caspase-12. Indeed, in NCX3-silenced neurons, Aß(1-42) exposure hastened caspase-dependent apoptosis, thus reinforcing neuronal cell death. These results suggest that Aß(1-42), through Ca(2+)-dependent calpain activation, generates a hyperfunctional form of NCX3 that, by increasing Ca(2+) content into ER, delays caspase-12 activation and thus neuronal death.

Na(+)/Ca (2+) exchange and the plasma membrane Ca(2+)-ATPase in ß-cell function and diabetes.

The rat pancreatic ß-cell expresses two splice variants of the Na+/Ca(2+) exchanger 1 (NCX1) and six splice variants of the plasma membrane Ca(2+)-ATPase (PMCA). In the ß-cell, Na(+)/Ca(2+) exchange displays a high capacity, contributes to both Ca(2+) outflow and influx and participates to the control of insulin release. Gain of function studies show that overexpression of NCX1 or PMCA2 leads to endoplasmic reticulum (ER) Ca(2+) depletion with subsequent ER stress, decrease in ß-cell proliferation and ß-cell death by apoptosis. Interestingly, chronic exposure to cytokines or high free fatty acids concentration also induces ER Ca(2+) depletion and ß-cell death in diabetes. Loss of function studies shows, on the contrary, that heterozygous inactivation of NCX1 (Ncx1 ( +/- )) leads to an increase in ß-cell function (insulin production and release) and a fivefold increase in both ß-cell mass and proliferation. The mutation also increases ß-cell resistance to hypoxia, and Ncx1 ( +/- ) islets show a four to seven times higher rate of diabetes cure than Ncx1 ( +/+ ) islets when transplanted in diabetic animals. Thus, downregulation of the Na(+)/Ca(2+) exchanger leads to various changes in ß-cell function that are opposite to the major abnormalities seen in diabetes. In addition, the ß-cell, which is an excitable cell, includes the mutually exclusive exon B in the alternative splicing region of NCX1, which confers a high sensitivity of its NCX splice variants (NCX1.3 & 1.7) to the inhibitory action of compounds like KB-R7943. This provides a unique model for the prevention and treatment of ß-cell dysfunction in diabetes and following islet transplantation.

Genetically modified mice as a strategy to unravel the role played by the Na(+)/Ca (2+) exchanger in brain ischemia and in spatial learning and memory deficits.

Because no isoform-specific blocker of NCX has ever been synthesized, a more selective strategy to identify the role of each antiporter isoform in the brain was represented by the generation of knockout and knockin mice for the different isoforms of the antiporter.Experiments performed in NCX2 and NCX3 knockout mice provided evidence that these two isoforms participate in spatial learning and memory consolidation, although in an opposite manner. These new data from ncx2-/- and ncx3-/- mice may open new experimental avenues for the development of effective therapeutic compounds that, by selectively inhibiting or activating these molecular targets, could treat patients affected by cognitive impairment including Alzheimer's, Parkinson's, Huntington's diseases, and infarct dementia.More importantly, knockout and knockin mice also provided new relevant information on the role played by NCX in maintaining the intracellular Na(+) and Ca(2+) homeostasis and in protecting neurons during brain ischemia. In particular, both ncx2-/- and ncx3-/- mice showed an increased neuronal vulnerability after the ischemic insult induced by transient middle cerebral artery occlusion.As the ubiquitous deletion of NCX1 brings about to an early death of embryos because of a lack of heartbeat, this strategy could not be successfully pursued. However, information on the role of NCX1 in normal and ischemic brain could be obtained by developing conditional knockout mice lacking NCX1 in the brain. Preliminarily results obtained in these conditional mice suggest that also NCX1 protects neurons from ischemic cell death.Overall, the use of genetic-modified mice for NCX1, NCX2, and NCX3 represents a fruitful strategy to characterize the physiological role exerted by NCX in CNS and to identify the isoforms of the antiporter as potential molecular targets for therapeutic intervention in cerebral ischemia.

Na+ -Ca2+ exchanger (NCX3) knock-out mice display an impairment in hippocampal long-term potentiation and spatial learning and memory.

Long-term potentiation (LTP) depends on the coordinated regulation of an ensemble of proteins related to Ca(2+) homeostasis, including Ca(2+) transporters. One of the major players in the regulation of intracellular Ca(2+) ([Ca(2+)](i)) homeostasis in neurons is the sodium/calcium exchanger (NCX), which represents the principal mechanism of Ca(2+) clearance in the synaptic sites of hippocampal neurons. Because NCX3, one of the three brain isoforms of the NCX family, is highly expressed in the hippocampal subfields involved in LTP, we hypothesized that it might represent a potential candidate for LTP modulation. To test this hypothesis, we first examined the effect of ncx3 gene ablation on NCX currents (I(NCX)) and Ca(2+) homeostasis in hippocampal neurons. ncx3(-/-) neurons displayed a reduced I(NCX), a higher basal level of [Ca(2+)](i), and a significantly delayed clearance of [Ca(2+)](i) following depolarization. Furthermore, measurement of field EPSPs, recorded from the CA1 area, revealed that ncx3(-/-) mice had an impaired basal synaptic transmission. Moreover, hippocampal slices from ncx3(-/-) mice exhibited a worsening in LTP compared with congenic ncx3(+/+). Consistently, immunohistochemical and immunoblot analysis indicated that in the hippocampus of ncx3(-/-) mice both Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) expression and the phosphoCaMKIIα/CaMKIIα ratio were significantly reduced compared with ncx3(+/+). Interestingly, ncx3(-/-) mice displayed a reduced spatial learning and memory performance, as revealed by the novel object recognition, Barnes maze, and context-dependent fear conditioning assays. Collectively, our findings demonstrate that the deletion of the ncx3 gene in mice has detrimental consequences on basal synaptic transmission, LTP regulation, spatial learning, and memory performance.

Sodium-calcium exchanger-3 regulates pain "wind-up": From human psychophysics to spinal mechanisms.

Repeated application of noxious stimuli leads to a progressively increased pain perception; this temporal summation is enhanced in and predictive of clinical pain disorders. Its electrophysiological correlate is "wind-up," in which dorsal horn spinal neurons increase their response to repeated nociceptor stimulation. To understand the genetic basis of temporal summation, we undertook a GWAS of wind-up in healthy human volunteers and found significant association with SLC8A3 encoding sodium-calcium exchanger type 3 (NCX3). NCX3 was expressed in mouse dorsal horn neurons, and mice lacking NCX3 showed normal, acute pain but hypersensitivity to the second phase of the formalin test and chronic constriction injury. Dorsal horn neurons lacking NCX3 showed increased intracellular calcium following repetitive stimulation, slowed calcium clearance, and increased wind-up. Moreover, virally mediated enhanced spinal expression of NCX3 reduced central sensitization. Our study highlights Ca2+ efflux as a pathway underlying temporal summation and persistent pain, which may be amenable to therapeutic targeting.

Plasma membrane Ca2+-ATPase overexpression depletes both mitochondrial and endoplasmic reticulum Ca2+ stores and triggers apoptosis in insulin-secreting BRIN-BD11 cells.

Ca(2+) may trigger apoptosis in ß-cells. Hence, the control of intracellular Ca(2+) may represent a potential approach to prevent ß-cell apoptosis in diabetes. Our objective was to investigate the effect and mechanism of action of plasma membrane Ca(2+)-ATPase (PMCA) overexpression on Ca(2+)-regulated apoptosis in clonal ß-cells. Clonal ß-cells (BRIN-BD11) were examined for the effect of PMCA overexpression on cytosolic and mitochondrial [Ca(2+)] using a combination of aequorins with different Ca(2+) affinities and on the ER and mitochondrial pathways of apoptosis. ß-cell stimulation generated microdomains of high [Ca(2+)] in the cytosol and subcellular heterogeneities in [Ca(2+)] among mitochondria. Overexpression of PMCA decreased [Ca(2+)] in the cytosol, the ER, and the mitochondria and activated the IRE1α-XBP1s but inhibited the PRKR-like ER kinase-eIF2α and the ATF6-BiP pathways of the ER-unfolded protein response. Increased Bax/Bcl-2 expression ratio was observed in PMCA overexpressing ß-cells. This was followed by Bax translocation to the mitochondria with subsequent cytochrome c release, opening of the permeability transition pore, and apoptosis. In conclusion, clonal ß-cell stimulation generates microdomains of high [Ca(2+)] in the cytosol and subcellular heterogeneities in [Ca(2+)] among mitochondria. PMCA overexpression depletes intracellular [Ca(2+)] stores and, despite a decrease in mitochondrial [Ca(2+)], induces apoptosis through the mitochondrial pathway. These data open the way to new strategies to control cellular Ca(2+) homeostasis that could decrease ß-cell apoptosis in diabetes.

Targeted disruption of Na+/Ca2+ exchanger 3 (NCX3) gene leads to a worsening of ischemic brain damage.

Na+/Ca+ exchanger 3 (NCX3), one of the three isoforms of the NCX family, is highly expressed in the brain and is involved in the maintenance of intracellular Na+ and Ca2+ homeostasis. Interestingly, whereas the function of NCX3 under physiological conditions has been determined, its role under anoxia is still unknown. To assess NCX3 role in cerebral ischemia, we exposed ncx3-/- mice to transient middle cerebral artery occlusion followed by reperfusion. In addition, to evaluate the effect of ncx3 ablation on neuronal survival, organotypic hippocampal cultures and primary cortical neurons from ncx3-/- mice were subjected to oxygen glucose deprivation (OGD) plus reoxygenation. Here we report that ncx3 gene suppression leads to a worsening of brain damage after focal ischemia and to a massive neuronal death in all the hippocampal fields of organotypic cultures as well as in cortical neurons from ncx3-/- mice exposed to OGD plus reoxygenation. In addition, in ncx3-/- cortical neurons exposed to hypoxia, NCX currents, recorded in the reverse mode of operation, were significantly lower than those detected in ncx3+/+. From these results, NCX3 protein emerges as a new molecular target that may have a potential therapeutic value in modulating cerebral ischemia.

Effects of plasma membrane Ca(2+)-ATPase overexpression upon D-glucose metabolism in insulin-producing BRIN-BD11 cells.

In order to investigate the possible link between PMCA (plasma-membrane Ca(2+)-ATPase) activity and D-glucose catabolism in insulin-producing cells, BRIN-BD11 cells were transfected with two isoforms of PMCA2. Transfection of insulin-producing BRIN-BD11 cells with PMCA2yb and PMCA2wb was documented by RT-PCR (reverse transcription-PCR), Western blot analysis, indirect immunofluorescence microscopy and (45)Ca(2+) uptake by microsomes. In the transfected cells, the overexpression of PMCA coincided with three major anomalies of D-glucose metabolism, namely a lower rate of D-[5-(3)H]glucose utilization prevailing at a low extracellular concentration of D-glucose (1.1 mM), a low ratio between D-[U-(14)C]oxidation and D-[5-(3)H]glucose utilization prevailing at a high extracellular glucose concentration (16.7 mM), and a high ratio between the net generation of (14)C-labelled acidic metabolites and amino acids and that of (3)H(2)O from D-[5-(3)H]glucose. These anomalies resulted in a decreased estimated rate of ATP generation (linked to the catabolism of the hexose) and a lowered ATP cell content, whether at low or high extracellular D-glucose concentrations. The net uptake of (45)Ca(2+) by intact cells was also decreased in the transfected cells, but to a greater extent than can apparently be attributed to the change in the ATP-generation rate. These findings document the relevance of PMCA activity to both D-glucose metabolism and Ca(2+) handling in insulin-producing cells, with emphasis on the key role of both cytosolic and mitochondrial Ca(2+) concentrations in the regulation of D-glucose catabolism. They also reveal that overexpression of PMCA leads, in insulin-producing cells, to an imbalance between ATP generation and consumption.

The Na+/Ca2+ exchanger and the Plasma Membrane Ca2+-ATPase in ß-cell function and diabetes.

The rat pancreatic ß-cell expresses 6 splice variants of the Plasma Membrane Ca2+-ATPase (PMCA) and two splice variants of the Na+/Ca2+ exchanger 1 (NCX1). In the ß-cell Na+/Ca2+ exchange displays a high capacity, contributes to both Ca2+ outflow and influx and participates to the control of insulin release. Gain of function studies show that overexpression of PMCA2 or NCX1 leads to endoplasmic reticulum (ER) Ca2+ depletion with subsequent ER stress, decrease in ß-cell proliferation and ß-cell death by apoptosis. Loss of function studies show, on the contrary, that heterozygous inactivation of NCX1 (Ncx1+/-) leads to an increase in ß-cell function and a 5 fold increase in both ß-cell mass and proliferation. The mutation also increases ß-cell resistance to hypoxia, and Ncx1+/- islets show a 2-4 times higher rate of diabetes cure than Ncx1+/+ islets when transplanted in diabetic animals. Thus, down-regulation of the Na+/Ca2+ exchanger leads to various changes in ß-cell function that are opposite to the major abnormalities seen in diabetes. In addition, the ß-cell includes the mutually exclusive exon B in the alternative splicing region of NCX1, which confers a high sensitivity of its NCX splice variants (NCX1.3 & 1.7) to the inhibitory action of compounds like KBR-7943. Heterozygous inactivation of PMCA2 leads to apparented, though not completely similar results.These provide 2 unique models for the prevention and treatment of ß-cell dysfunction in diabetes and following islet transplantation.

Na+/Ca2+ Exchanger a Druggable Target to Promote ß-Cell Proliferation and Function.

An important feature of type 2 diabetes is a decrease in ß-cell mass. Therefore, it is essential to find new approaches to stimulate ß-cell proliferation. We have previously shown that heterozygous inactivation of the Na+/Ca2+ exchanger (isoform 1; NCX1), a protein responsible for Ca2+ extrusion from cells, increases ß-cell proliferation, mass, and function in mice. Here, we show that Ncx1 inactivation also increases ß-cell proliferation in 2-year-old mice and that NCX1 inhibition in adult mice by four small molecules of the benzoxyphenyl family stimulates ß-cell proliferation both in vitro and in vivo. NCX1 inhibition by small interfering RNA or small molecules activates the calcineurin/nuclear factor of activated T cells (NFAT) pathway and inhibits apoptosis induced by the immunosuppressors cyclosporine A (CsA) and tacrolimus in insulin-producing cell. Moreover, NCX1 inhibition increases the expression of ß-cell-specific genes, such as Ins1, Ins2, and Pdx1, and inactivates/downregulates the tumor suppressors retinoblastoma protein (pRb) and miR-193a and the cell cycle inhibitor p53. Our data show that Na+/Ca2+ exchange is a druggable target to stimulate ß-cell function and proliferation. Specific ß-cell inhibition of Na+/Ca2+ exchange by phenoxybenzamyl derivatives may represent an innovative approach to promote ß-cell regeneration in diabetes and improve the efficiency of pancreatic islet transplantation for the treatment of the disease.

Preconditioning, induced by sub-toxic dose of the neurotoxin L-BMAA, delays ALS progression in mice and prevents Na+/Ca2+ exchanger 3 downregulation.

Preconditioning (PC) is a phenomenon wherein a mild insult induces resistance to a later, severe injury. Although PC has been extensively studied in several neurological disorders, no studies have been performed in amyotrophic lateral sclerosis (ALS). Here we hypothesize that a sub-toxic acute exposure to the cycad neurotoxin beta-methylamino-L-alanine (L-BMAA) is able to delay ALS progression in SOD1 G93A mice and that NCX3, a membrane transporter able to handle the deregulation of ionic homeostasis occurring during ALS, takes part to this neuroprotective effect. Preconditioning effect was examined on disease onset and duration, motor functions, and motor neurons in terms of functional declines and severity of histological damage in male and female mice. Our findings demonstrate that a sub-toxic dose of L-BMAA works as preconditioning stimulus and is able to delay ALS onset and to prolong ALS mice survival. Interestingly, preconditioning prevented NCX3 downregulation in SOD1 G93A mice spinal cord, leading to an increased number of motor neurons associated to a reduced astrogliosis, and reduced the denervation of neuromuscular junctions observed in SOD1 G93A mice. These protective effects were mitigated in ncx3+/- mice. This study established for the first time an animal model of preconditioning in ALS and candidates NCX3 as a new therapeutic target.

Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3.

We produced and analyzed mice deficient for Na/Ca exchanger 3 (NCX3), a protein that mediates cellular Ca(2+) efflux (forward mode) or Ca(2+) influx (reverse mode) and thus controls intracellular Ca(2+) concentration. NCX3-deficient mice (Ncx3(-/-)) present a skeletal muscle fiber necrosis and a defective neuromuscular transmission, reflecting the absence of NCX3 in the sarcolemma of the muscle fibers and at the neuromuscular junction. The defective neuromuscular transmission is characterized by the presence of electromyographic abnormalities, including low compound muscle action potential amplitude, a decremental response at low-frequency nerve stimulation, an incremental response, and a prominent postexercise facilitation at high-frequency nerve stimulation, as well as neuromuscular blocks. The analysis of quantal transmitter release in Ncx3(-/-) neuromuscular junctions revealed an important facilitation superimposed on the depression of synaptic responses and an elevated delayed release during high-frequency nerve stimulation. It is suggested that Ca(2+) entering nerve terminals is cleared relatively slowly in the absence of NCX3, thereby enhancing residual Ca(2+) and evoked and delayed quantal transmitter release during repetitive nerve stimulation. Our findings indicate that NCX3 plays an important role in vivo in the control of Ca(2+) concentrations in the skeletal muscle fibers and at the neuromuscular junction.

Role of Na/Ca exchange and the plasma membrane Ca2+-ATPase in beta cell function and death.

Recent progresses concerning the Na/Ca exchanger (NCX) and the plasma membrane Ca2+-ATPase (PMCA) in the pancreatic beta cell are reviewed. The rat beta cell expresses two splice variants of NCX1 and six splice variants of the 4 PMCA isoforms. At the protein level, the most abundant forms are PMCA2 and PMCA3, providing the first evidence for the presence of these two isoforms in a non-neuronal tissue. Overexpression of NCX1 in an insulinoma cell line altered the initial rise in cytosolic-free Ca2+ concentration ([Ca2+]i) induced by membrane depolarization and the return of the [Ca2+]i to the baseline value on membrane repolarization, indicating that NCX contributes to both Ca2+ inflow and outflow in the beta cell. In contrast, overexpression of the PMCA markedly reduced the global rise in Ca2+ induced by membrane depolarization, indicating that the PMCA has a capacity higher than expected to extrude Ca2+. Glucose, the main physiological stimulus of insulin release from the beta cell, has opposite effect on NCX and PMCA transcription, expression and activity, inducing an increase in the case of NCX and a decrease in the case of the PMCA. This indicates that when exposed to glucose, the beta cell switches from a low-efficiency Ca2+ extruding mechanism, the PMCA, to a high-capacity system, the NCX, in order to better face the increase in Ca2+ inflow induced by the sugar. To our knowledge, this is the first demonstration of a reciprocal change in PMCA and NCX1 expression and activity in response to a given stimulus in any tissue.

Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells.

Cytokines and free radicals are mediators of beta-cell death in type 1 diabetes. Under in vitro conditions, interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma) induce nitric oxide (NO) production and apoptosis in rodent and human pancreatic beta-cells. We have previously shown, by microarray analysis of primary beta-cells, that IL-1beta + IFN-gamma decrease expression of the mRNA encoding for the sarcoendoplasmic reticulum pump Ca(2+) ATPase 2b (SERCA2b) while inducing expression of the endoplasmic reticulum stress-related and proapoptotic gene CHOP (C/EBP [CCAAT/enhancer binding protein] homologous protein). In the present study we show that cytokine-induced apoptosis and necrosis in primary rat beta-cells and INS-1E cells largely depends on NO production. IL-1beta + IFN-gamma, via NO synthesis, markedly decreased SERCA2b protein expression and depleted ER Ca(2+) stores. Of note, beta-cells showed marked sensitivity to apoptosis induced by SERCA blockers, as compared with fibroblasts. Cytokine-induced ER Ca(2+) depletion was paralleled by an NO-dependent induction of CHOP protein and activation of diverse components of the ER stress response, including activation of inositol-requiring ER-to-nucleus signal kinase 1alpha (IRE1alpha) and PRK (RNA-dependent protein kinase)-like ER kinase (PERK)/activating transcription factor 4 (ATF4), but not ATF6. In contrast, the ER stress-inducing agent thapsigargin triggered these four pathways in parallel. In conclusion, our results suggest that the IL-1beta + IFN-gamma-induced decrease in SERCA2b expression, with subsequent depletion of ER Ca(2+) and activation of the ER stress pathway, is a potential contributory mechanism to beta-cell death.

Plasma membrane Ca(2+)-ATPase overexpression reduces Ca(2+) oscillations and increases insulin release induced by glucose in insulin-secreting BRIN-BD11 cells.

In the mouse beta-cell, glucose generates large amplitude oscillations of the cytosolic-free Ca(2+) concentration ([Ca(2+)](i)) that are synchronous to insulin release oscillations. To examine the role played by [ Ca(2+)](i) oscillations in the process of insulin release, we examined the effect of plasma membrane Ca(2+)-ATPase (PMCA) overexpression on glucose-induced Ca(2+) oscillations and insulin release in BRIN-BD11 cells. BRIN-BD11 cells were stably transfected with PMCA2wb. Overexpression could be assessed at the mRNA and protein level, with appropriate targeting to the plasma membrane assessed by immunofluorescence and the increase in PMCA activity. In response to K(+), overexpressing cells showed a markedly reduced rise in [Ca(2+)](i). In response to glucose, control cells showed large amplitude [Ca(2+)](i) oscillations, whereas overexpressing cells showed markedly reduced increases in [Ca(2+)](i) without such large oscillations. Suppression of [Ca(2+)](i) oscillations was accompanied by an increase in glucose metabolism and insulin release that remained oscillatory despite having a lower periodicity. Hence, [Ca(2+)] (i) oscillations appear unnecessary for glucose-induced insulin release and may even be less favorable than a stable increase in [ Ca(2+)](i) for optimal hormone secretion. [Ca(2+)](i) oscillations do not directly drive insulin release oscillations but may nevertheless intervene in the fine regulation of such oscillations.

Na/Ca exchanger overexpression induces endoplasmic reticulum-related apoptosis and caspase-12 activation in insulin-releasing BRIN-BD11 cells.

Ca(2+) may trigger programmed cell death (apoptosis) and regulate death-specific enzymes. Therefore, the development of strategies to control Ca(2+) homeostasis may represent a potential approach to prevent or enhance cell apoptosis. To test this hypothesis, the plasma membrane Na/Ca exchanger (NCX1.7 isoform) was stably overexpressed in insulin-secreting tumoral cells. NCX1.7 overexpression increased apoptosis induced by endoplasmic reticulum (ER) Ca(2+)-ATPase inhibitors, but not by agents increasing intracellular calcium concentration ([Ca(2+)](i)), through the opening of plasma membrane Ca(2+)-channels. NCX1.7 overexpression reduced the rise in [Ca(2+)](i) induced by all agents, depleted ER Ca(2+) stores, sensitized the cells to Ca(2+)-independent proapoptotic signaling pathways, and reduced cell proliferation by approximately 40%. ER Ca(2+) stores depletion was accompanied by the activation of the ER-specific caspase (caspase-12), and the activation was enhanced by ER Ca(2+)-ATPase inhibitors. Hence, Na/Ca exchanger overexpression, by depleting ER Ca(2+) stores, triggers the activation of caspase-12 and increases apoptotic cell death. By increasing apoptosis and decreasing cell proliferation, overexpression of Na/Ca exchanger may represent a new potential approach in cancer gene therapy. On the other hand, our results open the way to the development of new strategies to control cellular Ca(2+) homeostasis that could, on the contrary, prevent the process of apoptosis that mediates, in part, beta-cell autoimmune destruction in type 1 diabetes.