Na/Ca exchange and Ca2+ homeostasis in the pancreatic beta-cell.

Recent knowledge concerning the Na/Ca exchanger (NCX) in the pancreatic beta-cell is reviewed. The beta-cell expresses various NCX1 splice variants in a species specific pattern (NCX1.3 and 1.7 in the rat, NCX1.2, 1.3 and 1.7 in the mouse), in variable and different proportions. In the rat beta-cell, the exchanger displays a quite high capacity, accounts for about 70% of Ca2+ extrusion and participates to Ca2+ inflow during membrane depolarization. In the mouse, however, the contribution of the exchanger to Ca2+ extrusion is more modest, and to Ca2+ inflow less evident. The exchanger has a stoichiometry of 3 Na+ for 1 Ca2+, is electrogenic, and displays a reversal potential at -20 mV. Although being of low magnitude, the current generated by the exchanger shapes glucose-induced beta-cell electrical activity and intracellular Ca2+ oscillations. For instance, a lower Na/Ca exchange activity (and subsequent inward current) in the mouse than in the rat, explains why the mouse beta-cell displays cyclic oscillations of the membrane potential, while the rat displays a staircase increase in membrane potential without such oscillations. In addition of being of importance in cell signalling, intracellular Ca2+ may also trigger apoptosis. For instance, overexpression of the exchanger increases Ca2+-dependent and -independent beta-cell death by apoptosis, a phenomenon resulting from the depletion of ER Ca2+-stores with subsequent activation of caspase-12. Na/Ca exchange overexpression also reduces beta-cell growth. Hence, the Na/Ca exchanger is a versatile system that appears to play an important role in the function, growth and demise of the beta-cell.

Novel inhibitors of the sodium-calcium exchanger: benzene ring analogues of N-guanidino substituted amiloride derivatives.

A series of N-guanidino substituted 2,4-diamino-5-carbonylguanidine molecules related to amiloride were synthesised and evaluated for their ability to inhibit the sodium-calcium exchanger in rat insulinoma cells (RINm5F) and human platelets. Specific chemical pathways were used to prepare the benzene derivatives designed as bioisosteric analogues of the pyrazine derivatives of amiloride. Several so-called 'simplified analogues', where some substituents of amiloride were omitted or replaced, were also prepared and included in the biological evaluation. The inhibitory potency of the sodium-calcium exchanger was screened on both cell types by measuring their effect on 45Ca(2+) uptake. Among the most active compounds, N-(2-amino-5-chloro-4-nitrobenzoyl)-N'-(1-naphtylmethyl)guanidine (IC(50)=3.4 microM) was found more active than amiloride (IC(50)=690 microM) and 3,4-dichlorobenzamil (IC(50)=15.2 microM), the reference inhibitor.

A new Na/Ca exchanger splicing pattern identified in situ leads to a functionally active 70kDa NH(2)-terminal protein.

The Na/Ca exchanger (NCX) is an ubiquitous transporter that plays an important role in regulating cellular Ca(2+) balance. On gel electrophoresis, the NCX1 protein migrates as two major bands of 120 and 70kDa. While the 120kDa is thought to represent the native protein, the nature of the 70kDa protein remains unclear. In this report, we describe a new NCX1 splicing pattern, identified during the cloning of NCX1 isoforms from human eye. The insertion of a newly identified sequence upstream exons B and D of the NCX1.3 isoform, generates a stop codon in frame with the NCX1 coding sequence, that should lead to a truncated Na/Ca exchanger (that we called NCX1.33) comprising only the N-terminal portion of the exchanger and a shortened intracellular loop. Insulin-secreting cells were stably transfected with NCX1.33. Overexpression was assessed at the mRNA and protein level, the truncated exchanger migrating as a70kDa band. Appropriate targeting to the plasma membrane was assessed by microfluorescence and by the increase in Na/Ca exchange activity. The results of the present study constitute a clear piece of evidence indicating that the Na/Ca exchanger 70kDa protein corresponds to the N-terminal portion of the exchanger, and is functionally active.

NCX1 Na/Ca exchanger splice variants in pancreatic islet cells.

In the rat pancreatic beta-cell, Na/Ca exchange displays a quite high capacity. The cell is equipped with two alternatively spliced Na/Ca exchanger-1 (NCX1) isoforms, namely NCX1.3 and NCX1.7. To examine the existence of a possible functional difference between these splice variants, they were cloned, together with the heart variant NCX1.1, and expressed in human embryonic kidney-293 (HEK293) and Chinese hamster ovary (CHO) cells. In these two systems, the three splice variants showed a comparable level of intracellular Na+ (Na+(i))-dependent extracellular Ca2+(Ca2+(o)) uptake. Different levels of NCX1.3 and NCX1.7 transcripts were found in four rodent species, together with a marked interspecies difference in Na/Ca exchange activity. Three additional splice variants were found (NCX1.2, NCX1.9 and NCX1.13) in guinea-pigs, hamsters and mice, again in different proportions. Our data provide evidence for the activity of the three NCX1 splice variants. They also show the existence of a differential and species-specific transcription pattern of NCX1 gene products in pancreatic islet cells.

Expression of multiple plasma membrane Ca(2+)-ATPases in rat pancreatic islet cells.

When stimulated by glucose, the pancreatic beta-cell displays large oscillations of intracellular free Ca2+ concentration ([Ca2+]i). To control [Ca2+]i, the beta-cell must be equipped with potent mechanisms for Ca2+ extrusion. We studied the expression of the plasma membrane Ca(2+)-ATPases (PMCA) in three insulin secreting preparations (a pure beta-cell preparation, RINm5F cells and pancreatic islet cells), using reverse-transcribed PCR, RNase protection assay and Western blotting. The four main isoforms, PMCA1, PMCA2, PMCA3 and PMCA4 were expressed in the three preparations. Six alternative splice mRNA variants, characterized at splice sites A, B and C were detected in the three preparations (rPMCA1xb, 2yb, 2wb, 3za, 3zc, 4xb), plus two additional variants in pancreatic islet cells (PMCA4za, 1xkb). The latter variant corresponded to a novel variant of rat PMCA1 gene lacking the exon coding for the 10th transmembrane segment, at splice site B. At the mRNA and protein level, five variants predominated (1xb, 2wb, 3za, 3zc, 4xb), whilst one additional isoform (4za), predominated at the protein level only. This provides the first evidence for the presence of PMCA2 and PMCA3 isoforms at the protein level in non-neuronal tissue. Hence, the pancreatic beta-cell is equipped with multiple PMCA isoforms with possible differential regulation, providing a full range of PMCAs for [Ca2+]i regulation.

Contribution of Na/Ca exchange to Ca2+ outflow and entry in the rat pancreatic beta-cell: studies with antisense oligonucleotides.

To characterize the role played by Na/Ca exchange in the pancreatic beta-cell, phosphorothioated antisense oligonucleotides (AS-oligos) were used to knock down the exchanger in rat pancreatic beta-cells. Na/Ca exchange activity was evaluated by measuring cytosolic free Ca2+ concentration ([Ca2+]i) in single cells using fura-2. Exposure of beta-cells to 500 nmol/l of the AS-oligos for 24 h inhibited Na/Ca exchange activity by approximately 77%. In contrast, control oligonucleotides (scrambled and mismatched) did not affect Na/Ca exchange activity. In AS-oligo-treated cells, the increase in [Ca2+]i induced by membrane depolarization (K+ or the hypoglycemic sulfonylurea, tolbutamide) was reduced by 28 or 40%, respectively. Likewise, the rate of [Ca2+]i decrease after K+ or tolbutamide removal was reduced by 72 or 40%, respectively. AS-oligos treatment also abolished the nifedipine-resistant increase in [Ca2+]i induced by K+ and profoundly altered the oscillatory or sustained increases in [Ca2+]i induced by 11.1 mmol/l glucose. The present study shows that AS-oligos may specifically inhibit Na/Ca exchange in rat pancreatic beta-cells. In those cells, Na/Ca exchange appears to mediate Ca2+ entry in response to membrane depolarization and to be responsible for up to 70% of Ca2+ removal from the cytoplasm upon membrane repolarization.

The proximal interferon-stimulated response elements are essential for interferon responsiveness: a promoter analysis of the antiviral MxA gene.

Interferon (IFN)-inducible human MxA protein mediates resistance against influenza and several other RNA viruses. The MxA gene is under the control of type I IFN and, in certain cell types, is also directly activated by viruses. Here we show that in human macrophages, MxA mRNA levels are upregulated by very low doses of IFN-alpha in a dose-dependent manner. A similar, albeit much weaker, dose-dependent induction was seen with IFN-gamma. The induction was rapid and independent of protein synthesis. Interleukin-6 (IL-6) or tumor necrosis factor-alpha (TNF-alpha) did not influence MxA mRNA levels alone or in combination with IFNs, in spite of the presence of putative response elements of these cytokines in the MxA promoter. We show that the promoter of the MxA gene contains two functional IFN-stimulated response elements (ISRE) near the transcription start site and one homologous ISRE-like element, which is apparently nonfunctional, further upstream. The two proximal ISRE sites are essential for IFN-alpha-induced transcription and appear to be binding sites for IFN-stimulated gene factor 3 complex. In addition, EMSA and DNAse I footprinting analysis demonstrated that Spl binds with high affinity to a region encompassing nucleotides -25 and -50 and, thus, may provide means of interaction with the basal transcriptional machinery.

Identification, expression pattern and potential activity of Na/Ca exchanger isoforms in rat pancreatic B-cells.

In the pancreatic B-cell, Na/Ca exchange displays a quite high capacity and participates in the control of cytosolic free Ca2+ concentration. The Na/Ca exchanger was recently cloned in various tissues. Two genes coding for two different exchangers (NCX1 and NCX2) have been identified and evidence for several isoforms for NCX1 shown. To characterize the isoform(s) expressed in pancreatic B-cells, a RT-PCR analysis was performed on mRNA from rat pancreatic islets, purified B-cells and insulinoma B-cells (RINm5F cells). PCR amplification did not yield the expected NCX2 DNA fragment but yielded 2 NCX1 bands, corresponding to NaCa3 and NaCa7, in the three preparations. NaCa3 and NaCa7 were equally expressed in pancreatic islets and purified B-cells. In RINm5F cells, NaCa3 expression did not differ from that in islet and purified B-cells but NaCa7 was 3 times less expressed. This lower expression was accompanied by a 3 times lower Na/Ca exchange activity in RINm5F cells compared to islet cells. Our data indicate the existence of 2 NCX1 isoforms but not of NCX2 in pancreatic B-cells. The difference in both the expression patterns of NCX1 isoforms and the activity of Na/Ca exchange in islet cells and RINm5F cells is compatible with a difference in activity between NaCa3 and NaCa7.

Inhibition of Na/Ca exchange by Phe-Met-Arg-Phe-NH2 (FMRFa)-related peptides in intact rat pancreatic B-cells.

Phe-Met-Arg-Phe-NH2 (FMRFa)-related peptides were recently shown to inhibit Na/Ca exchange in cardiac sarcolemmal vesicles. In the present study, we examined the effects of FMRFa-related peptides on Na/Ca exchange in intact (pancreatic B) cells. At 2.8 mM glucose, FMRFa-related peptides only weakly inhibited Na/Ca exchange although their effect was more marked under depolarizing conditions. The peptides blocked neither the Na/K-ATPase nor Ca2+ channels but slightly reduced membrane K+ permeability. Our data indicate that FMRFa-related peptides are weak and non-specific inhibitors of Na/Ca exchange in intact B cells. The data do not confirm the view that the peptides may exert some of their physiological modulatory role by inhibiting Na/Ca exchange.

Inhibition of Na/Ca exchange stimulates insulin release from isolated rat pancreatic islets.

Na/Ca exchange was recently shown to regulate cytosolic free Ca2+ concentration ([Ca2+]i) in the pancreatic B-cell. The aim of the present study was to provide direct evidence that inhibition of the activity of the exchange may also increase insulin release. In the presence of extracellular Na+, caffeine stimulated 45Ca outflow but did not increase insulin release from islets perifused in the presence of 2.8 mM glucose. By contrast, in the absence of extracellular Na+, caffeine almost failed to increase 45Ca outflow and reversibly stimulated insulin release despite the fact that the absence of extracellular Na+ per se reduced basal insulin release. Similar findings were observed in islets perifused at a higher glucose concentration (8.3 mM) except that, in the presence of extracellular Na+, caffeine more markedly increased 45Ca outflow and stimulated insulin release. Our data provide direct evidence that inhibition of Na/Ca exchange with resulting blockade of Ca2+ outflow may increase insulin release from the pancreatic B-cell under suitable experimental conditions.