Overexpression of the Na/Ca Exchanger Shapes Stimulus-Induced Cytosolic Ca²⁺ Oscillations in Insulin-Producing BRIN-BD11 Cells

The human pancreatic islet NCX1.7 cDNA was isolated from human insulinoma and cloned using the RT-PCR method as previously described (22).

BRIN-BD11 cells produced by electrofusion of normal rat pancreatic β-cells with RINm5F cells (31) were cultured in RPMI medium containing 11.1 mmol/l glucose supplemented with 10% FCS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10 μg/ml gentamicine (Life Technologies, Merelbeke, Belgium) in a humidified atmosphere of 5% CO₂ at 37°C.

Cells were plated at 3 × 10⁵ on a 35-mm-diameter plastic dish and incubated 24 h before transfection. BRIN-BD11 cells were then stably transfected with the mammalian expression vector pcDNA3(−) containing a 2.9-kb insert encoding for human pancreatic β-cell NCX1.7 by the use of LipofectAMINE (Life Technologies). Positive clones were selected through resistance against G418 (250 μg/ml; Life Technologies) and verified by RT-PCR, Western blot analysis, immunofluorescence, and Na/Ca exchange activity.

For RT-PCR, primers were designed to anneal to conserved sequences flanking the putative splicing area of NCX1. The sense primer 5′-CATTGGCATCATGGAGGT-3′ and the antisense primer 5′-TTTGCTGGTCAGTGGCTGCTTGTC-3′ correspond to nucleotides 1647–1664 and 2044–2067, respectively, based on rat heart sequence (GenBank accession no. x68191). The oligonucleotides were synthesized using the phosphoramidite method performed on an Applied Biosystems 394 synthesizer (Perkin Elmer, Zaventem, Belgium).

Total RNA was isolated from control and transfected BRIN-BD11 cells using the RNA Now method (Biogentex, Seabrooke, TX). RNA (1 μg) was reverse transcribed for 20 min at 42°C and 40 min at 37°C using a Superscript II kit (Life Technologies), with 25 μg/ml oligo(dT) primer and 2.5 μg/ml random primer (Promega), and triphosphate nucleosides (0.5 mmol/l each; Boehringer Mannheim, Brussels), in the buffer supplied by the manufacturer, in a total volume of 20 μl. RNA complementary to cDNA was removed using two units of Escherichia coli Rnase H (Boehringer Mannheim) for 20 min at 37°C. The medium was then diluted with 30 μl of 16 mmol/l EDTA, and the reaction was terminated by heating to 99°C for 5 min. PCR amplification was carried out as described elsewhere (20).

Plasma membranes were isolated from control BRIN-BD11 cells as well as transfected cells as previously described (22).

Western blot analysis was carried out as previously described (22).

Cells on coverslips were analyzed by indirect immunofluorescence microscopy 48–60 h after plating. After washing with a Tris-buffered saline (TBS) solution (20 mmol/l Tris, 137 mmol/l NaCl, pH 7.2), the cells were fixed for 20 min in 4% formaldehyde at pH 7.4 (4°C); washed with TBS; permeabilized with a solution containing 0.01% Triton X-100, 197 μmol/l MgCl₂, 19.5 μmol/l dithiothreitol, and 10% glycerol (pH 7.4, 4°C); washed twice with TBS; and incubated in a blocking buffer containing 1% horse serum (Vector Laboratories, Burlingame, CA) in TBS for 20 min. The coverslips were exposed for 1 h to the primary antibody (mouse monoclonal anti-canine Na⁺-Ca²⁺-Exchanger; SWant, Bellinzona, Switzerland) diluted 1/1,000 in TBS containing 1% BSA. Control cells were incubated in TBS-1% BSA buffer without antibody and washed three times with TBS. The cells were then treated with the secondary antibody (Alexa Fluor 594 goat anti-mouse IgG [H + L] conjugate; Molecular Probes, Eugene, OR) diluted 1/400 in TBS-1% BSA for 45 min and washed four times with TBS. The cells were incubated with 300 nmol/l DAPI solution (4′,6-diamino-2-phenylindole; Molecular Probes) and washed twice with TBS. The coverslips were mounted on slides using Vectashield (Vector Laboratories) and examined using an Axioplan microscope (Zeiss, Oberkochen, Germany) equipped with an HBO 100 W or XBO 100 W illuminator and photographed with a dual-mode cooled CCD C4880 camera (Hamamatsu, Hamamatsu, Japan).

For ⁴⁵Ca uptake, the media used to incubate BRIN-BD11 cells consisted in a Krebs-Ringer HEPES-buffered solution (pH 7.4, 37°C) with the following composition (in mmol/l): 135 NaCl, 1 CaCl₂, 5 KCl, 1 MgCl₂, and 10 HEPES/NaOH. Unless otherwise stated, the media were gassed with ambient air. In some experiments, NaCl was iso-osmotically replaced by sucrose (241 mmol/l; Merck, Darmstadt, Germany) and HEPES/NaOH by HEPES/KOH. For the measurement of Na⁺_i [intracellular Na⁺]-dependent ⁴⁵Ca uptake, the different media also contained glucose (2.8 mmol/l; Merck) and nifedipine (5 μmol/l; Calbiochem, La Jolla, CA). For the measurement of the effect of glucose, the media contained glucose (1.1 or 11.1 mmol/l) without nifedipine. The method used for the measurement of ⁴⁵Ca uptake has been described previously (10).

For the measurement of [Ca²⁺]_i, a drop of the cell suspension (20 μl) containing 50,000 cells was aliquoted onto round glass coverslips placed in Petri dishes and incubated at 37°C in a 5% CO₂/95% O₂ incubator. After 2 h, fresh culture medium was added to the Petri dishes. The cells were further incubated for 24 h at 37°C before use. The medium used to incubate or perfuse the islet cells consisted in a Krebs-Ringer bicarbonate-buffered solution (pH 7.4, 37°C) having the following composition (in mmol/l): 115 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 24 NaHCO₃, and 1.1 glucose. The medium was equilibrated against a mixture of O₂ (95%) and CO₂ (5%) at pH 7.4. For Na⁺-free solutions, NaCl was iso-osmotically replaced by choline chloride and NaHCO₃ by choline bicarbonate. To avoid cholinergic effects, the latter media contained atropine (10 μmol/l), which was also present in appropriate control solutions. The different media also contained, when required, 11.1 mmol/l glucose, 50 mmol/l KCl, 100 μmol/l tolbutamide, and 5 μmol/l diazoxide. Stock solution containing fura-2-AM was prepared in dimethyl sulfoxide. Cells were incubated with fura-2-AM ester (final concentration 2 μmol/l) during 60 min at 37°C in a Krebs-Ringer bicarbonate-buffered solution containing 1.1 mmol/l glucose. The coverslips were then transferred to a tissue chamber mounted on an inverted fluorescence microscope (Diaphot TDM; Nikon, Tokyo) for epifluorescence, as described previously (13).

Cells were plated on plastic dishes and used 24 h later. Whole-cell membrane currents were recorded at room temperature (22°C) with borosilicate glass patch electrodes (with tip resistances of 1–2 MΩ when filled with the internal solution) and connected to the head stage of an EPC7 patch clamp amplifier (List Electronics, Darmstadt, Germany) or an Axopatch 100A amplifier (Axon Instruments, Foster City, CA). Voltage clamp and data acquisition and storage were carried out using the Pulse software (r. 7.89; Heka Electronics, Lambrecht/Pfalz, Germany) or pClamp software (Axon Instruments).

Cell capacitance was obtained by integrating the current generated upon a short hyperpolarization from the holding potential of −70 to −80 mV. The amplitude of the Ca²⁺ current (I_Ca) induced by a given depolarization step was determined as the difference between the peak inward current and the current at the end of the step using Origin (Microcal, Northampton, MA).

The extracellular solution during measurements had the following composition (in mmol/l): 135 NaCl, 5.4 CsCl, 2.6 CaCl₂, 0.9 MgCl₂, 0.33 NaH₂PO₄, 10 HEPES, and 10 glucose, pH 7.4 (titrated with NaOH). The composition of the pipette solution was (in mmol/l): 130 Cs-glutamate, 25 CsCl, 1 MgCl₂, 1 EGTA, 5 Na₂ATP, 0.1 Na₂GTP, and 5 HEPES, pH 7.20 (titrated with CsOH).

Insulin release from BRIN-BD11 cells in tissue culture was determined using cell monolayers. The BRIN-BD11 cells were harvested with the aid of trypsin and were seeded into 24-multiwell plates at a density of 5 × 10⁵ per well and were allowed to attach overnight at 37°C. Standard RPMI-1640 tissue culture medium containing 11.1 mmol/l glucose, which was routinely used to culture the hybrid β-cells, was removed from each well and replaced with culture media containing 1.1, 11.1, and 33.3 mmol/l glucose, respectively. The cells were then incubated for 24 h, and aliquots of the culture media were removed from each separate well and stored at −20°C for subsequent insulin measurement by radioimmunoassay (12). Insulin output was expressed in terms of the number of cells in each well at the end of the experiment.

The results are expressed as means ± SE. The statistical significance of differences between data were assessed using the Student’s t test or ANOVA followed by Tukey’s post test.

BRIN-BD11 cells were stably transfected with a mammalian expression vector pcDNA3(−) containing an insert coding for the human pancreatic β-cell NCX1.7 exchanger (GenBank accession no. AF108388). A clone of cells expressing high levels of Na/Ca exchange activity (clone 8) was used for studies described here. However, similar results were observed with other clones (3, 4, 5, and 6).

The transcription and expression level of NCX1.7 protein in the transfected cells were determined by RT-PCR method, Western blot analysis, immunofluorescence, and Na/Ca exchange activity (Na⁺_i-dependent ⁴⁵Ca_o²⁺ [extracellular Ca²⁺] uptake by reverse Na/Ca exchange).

In a previous study, we identified two NCX1 isoforms (NCX1.3 and NCX1.7) in both rat pancreatic β-cells and RINm5F cells (20). With BRIN-BD11 cells being produced by electrofusion of normal rat pancreatic β-cells with RINm5F cells, we looked for the presence of these two NCX1 isoforms in BRIN-BD11 cells. Using primers designed to anneal to conserved sequences flanking the splicing area of NCX1, PCR amplification yielded two bands of 379 and 310 bp (Fig. 1A, lane 2) indicating the presence of exons BDF and BD characterizing NCX1.7 and NCX1.3 isoforms, respectively. In NCX1.7-transfected cells, PCR amplification also yielded two bands of identical molecular weight, with the upper NCX1.7 transcript being markedly enhanced (Fig. 1A, lane 3).

On gel electrophoresis, the Na/Ca exchanger migrates as a 120-kDa and 70-kDa protein, with the 120-kDa band being commonly accepted to represent the mature glycosylated protein and the 70-kDa protein representing a proteolytic fragment of the 120-kDa band (32). An immunoblot of membranes from control cells (Fig. 1B, lane 1) showed one single clear band at ∼120 kDa. An immunoblot of membranes from NCX1.7-transfected cells (Fig. 1B, lane 2) showed markedly increased amounts of both 120-kDa and 70-kDa bands. The newly expressed 120-kDa band migrated slightly more slowly than the native one, indicating a slight difference in protein processing. For the purpose of comparison, a comparative immunoblot of primary rat β-cell membranes and of nontransfected BRIN cells is shown in Fig. 1C. In rat β-cells, the exchanger migrated as a 120-kDa protein, the level of expression of the exchanger being comparable with that of nontransfected BRIN-BD11 cells.

The overexpression of NCX1.7 protein at the level of the plasma membrane of the transfected cells was visualized by immunofluorescence using a monoclonal antibody directed against the canine cardiac Na/Ca exchanger (Fig. 2). In these cells (e.g., clone 4 and 8), an intense staining predominantly located at the periphery of cells can be seen (Fig. 2, lower panels), whereas nontransfected cells only displayed a faint fluorescence at the periphery of the cells (Fig. 2, upper and right panel).

To obtain functional evidence of NCX1.7 overexpression, Ca²⁺ uptake and [Ca²⁺]_i changes induced by extracellular Na⁺ removal were examined (Figs. 3 and 4). In previous studies, we showed that this maneuver stimulated Ca²⁺ entry and [Ca²⁺]_i rise in pancreatic β-cells selectively by Na/Ca exchange (reverse mode) (10,11). Figure 3 illustrates the time courses of Na⁺_i-dependent Ca²⁺ uptake in control and NCX1.7-transfected cells. In both cell types, ⁴⁵Ca uptake displayed an initial and rapid phase followed by a plateau phase. However, the increase in ⁴⁵Ca uptake was more rapid and marked in transfected cells than in nontransfected cells. At the plateau phase (120 s), the increase in uptake was about two to three times larger in transfected cells than in nontransfected cells (P < 0.01). Figure 4 illustrates the effect of extracellular Na⁺ removal on [Ca²⁺]_i. In control cells, Na⁺ removal induced a modest and reversible [Ca²⁺]_i increase. In contrast, in NCX1.7-transfected cells, extracellular Na⁺ removal induced a more rapid, abrupt, and marked increase in [Ca²⁺]_i. Thus, at the peak, the increase in [Ca²⁺]_i was about four to five times larger in transfected cells than in nontransfected cells (P < 0.0001). In this series of experiments, like in others (Figs. 5–7), basal [Ca²⁺]_i did not differ between control and overexpressing cells and averaged 165 ± 8 nmol/l (n = 200).

Taken as a whole, these results support the view that Na/Ca exchange was functionally more active in NCX1.7-overexpressing cells than in control cells.

To evaluate the role played by Na/Ca exchange in Ca²⁺ homeostasis, we examined the effect of NCX1.7 exchanger overexpression on the changes in [Ca²⁺]_i induced by membrane depolarization. Figure 5 illustrates the effect of KCl (50 mmol/l) on [Ca²⁺]_i. In control cells, K⁺ induced a biphasic increase in [Ca²⁺]_i consisting in an initial peak followed by a plateau phase. The increase in [Ca²⁺]_i was rapidly reversible upon removal of K⁺ from the solution. In NCX1.7-overexpressing cells, several differences could be observed. First, K⁺ induced a more rapid increase in [Ca²⁺]_i. Thus, the maximum [Ca²⁺]_i was reached after 144 ± 5 and 112 ± 5 s in control and overexpressing cells, respectively (P < 0.0001). Likewise, whereas the rate of [Ca²⁺]_i increase averaged 10.1 ± 0.2 nmol · l⁻¹ · s⁻¹ (n = 94) in control cells, it averaged 17.8 ± 0.3 nmol · l⁻¹ · s⁻¹ in NCX1.7-overexpressing cells (n = 106, P < 0.0001). Second, the [Ca²⁺]_i observed at the steady state (before K⁺ removal) was higher in transfected cells (725 ± 17 nmol/l) than in control cells (610 ± 15 nmol/l, P < 0.0001). Last, the decrease in [Ca²⁺]_i seen on membrane repolarization was more rapid in overexpressing cells (7.0 ± 0.2 nmol · l⁻¹ · s⁻¹) than in control cells (5.2 ± 0.2 nmol · l⁻¹ · s⁻¹, P < 0.0001) and occurred about 30 s earlier.

To further examine the effect of NCX1.7 exchanger overexpression on Ca²⁺ handling in BRIN-BD11 cells, [Ca²⁺]_i changes evoked by 11.1 mmol/l glucose were investigated. This concentration of glucose elicits near-maximal stimulation of insulin secretion from BRIN-BD11 cells (31). Figure 6 illustrates typical individual [Ca²⁺]_i responses induced by glucose in control and NCX1.7-overexpressing cells (Fig. 6A and B, respectively). After a rapid increase in [Ca²⁺]_i, control cells responded to glucose by displaying large amplitude oscillations. In contrast, transfected cells responded by a delayed and more modest increase in [Ca²⁺]_i with small oscillations, which barely differed from those observed during the baseline. Although BRIN-BD11 cells showed high variability in their individual response, the mean response of all cells was calculated and is displayed in Fig. 6C. It is clear from the figure that a major difference was that while nontransfected cells showed clear oscillations, no such oscillations were seen in NCX1.7-transfected cells. At the steady state, the level of fluorescence was slightly lower in overexpressing cells than in control cells (173 ± 13 vs. 190 ± 14 nmol/l, P > 0.8).

Figure 7 shows typical individual [Ca²⁺]_i responses induced by 100 μmol/l tolbutamide (in the presence of 2.8 mmol/l glucose) (Fig. 7A and B) as well as the mean of the responses seen in control and NCX1.7-transfected cells (Fig. 7C). Like in the case of glucose, nontransfected cells displayed clear oscillations; overexpressing cells did not. Again in Fig. 7C, it can be seen that although control cells showed clear oscillations, the transfected cells showed a staircase increase in [Ca²⁺]_i without such distinct oscillations.

To exclude any influence of NCX1.7 overexpression on voltage-sensitive I_Ca, I_Ca was measured using the patch clamp technique. The holding potential was set at −70 mV, and the command voltage consisted of steps, given every 3 min, from the holding potential to a preconditioning level at −40 mV for 0.5 s (to inactivate voltage-dependent Na⁺ currents), followed by a 6- to 8-s depolarization to 0 mV to activate the L-type Ca²⁺ current. Round cells were used in both groups and were of the same size (membrane capacitance 17.7 ± 1.0 vs. 19.2 ± 1.6 pF in control and transfected cells, respectively). Gigaohm seal resistances were also similar (4.0 ± 1.1 vs. 3.5 ± 0.6 GΩ in control and transfected cells, respectively). The current at 0 mV could be suppressed by nifedipine (10 μmol/l), indicating that it was due to L-type Ca²⁺ channels. Our preliminary data (not presented) showed that I_Ca inactivates over a few seconds and recovers from inactivation slowly. Figure 8A illustrates typical recordings obtained in a control and in a transfected cell. Both currents decayed from their peak value, with a half-time of 724 and 868 ms, respectively. Pooled data of peak current amplitudes and half-time of decay from nine control and nine transfected cells are given in Fig. 8B and C, respectively. Although the current amplitude tended to be larger in transfected cells (−8.00 ± 1.7 pA/pF), the difference with control cells (−5.7 ± 0.8 pA/pF) did not reach statistical significance (P > 0.20). Similarly, there was no significant difference between the half-time of inactivation between the two groups of cells (1,051 ± 158 vs. 1,000 ± 178 ms in control and transfected cells, respectively).

If the absence of large amplitude [Ca²⁺]_i oscillations in overexpressing cells results from an increased Na/Ca exchange depolarizing current, then this absence should be reversed by membrane hyperpolarization. Figure 9 shows that this is indeed the case. Thus, diazoxide (2.5–10 μmol/l), which hyperpolarizes the plasma membrane by opening ATP-dependent K⁺ channels (33), restored glucose-induced large amplitude [Ca²⁺]_i oscillations in NCX1.7-overexpressing cells.

⁴⁵Ca uptake was measured over 90 min of incubation in the presence of glucose (1.1 and 11.1 mmol/l, Fig. 10A). In nontransfected cells, glucose induced a modest increase in ⁴⁵Ca uptake. In overexpressing cells, basal ⁴⁵Ca uptake (at 1.1 mmol/l glucose) was about 100% higher than that in nontransfected cells (P < 0.001), and glucose induced a six times larger increase in ⁴⁵Ca uptake than in nontransfected cells (P < 0.001).

Insulin release was measured over a 24-h static incubation in RPMI-1640 tissue culture medium in the presence of 1.1 or 11.1 mmol/l glucose. In nontransfected cells, 11.1 mmol/l glucose induced a two- to threefold increase in insulin release (P < 0.05, Fig. 10B). In NCX1.7 transfected cells, basal insulin release, measured at 1.1 mmol/l glucose, was markedly increased (P < 0.001), and the increase in insulin release induced by 11.1 mmol/l glucose was about 10 times larger than that in nontransfected cells (P < 0.01, Fig. 10B). To examine the extent to which this increase in insulin release could be related to the overexpression of the exchanger, the effect of glucose on insulin release was examined in four other clones (clones 3–6). However, none of the latter clones showed an increase in the insulinotropic action of glucose, suggesting that the potentiating effect on glucose-induced insulin release seen in clone 8 was not due to Na/Ca exchanger overexpression.

In the present work, we successfully and stably overexpressed one of the pancreatic β-cell Na/Ca exchanger splice variants (NCX1.7) in insulin-producing BRIN-BD11 cells. The overexpression could be assessed at both the mRNA and protein level. The overexpressed protein was appropriately targeted to the plasma membrane, as shown by microfluorescence but also by the increase in Na/Ca exchange activity observed in transfected cells.

The Na/Ca exchanger works predominantly as a Ca²⁺ extrusion mechanism (forward mode) but may also reverse (reverse mode) under physiological conditions, for example, upon membrane depolarization or in response to an increase in intracellular Na⁺ concentration (13,34). The present data clearly show that both modes of activity were increased in NCX1.7-overexpressing cells. The increase in forward mode activity was best evidenced in K⁺ experiments, where the decrease in [Ca²⁺]_i seen on membrane repolarization was about 30 s earlier and faster in overexpressing than in control cells. The increase in reverse Na/Ca exchange activity of our clone was also obvious, with the rise in ⁴⁵Ca uptake and [Ca²⁺]_i induced by the removal of extracellular Na⁺ being more marked in overexpressing cells than in control cells. This increase in reverse Na/Ca exchange activity was also evident from the K⁺ experiments, in which a more rapid rise (about twofold) in [Ca²⁺]_i was observed on membrane depolarization. Because the Ca²⁺ current was unchanged in overexpressing cells compared with control cells, the accelerated rise in [Ca²⁺]_i can be attributed to an increase in reverse Na/Ca exchange activity. Likewise, the higher [Ca²⁺]_i recorded at the steady state in the same experiments can also be taken as evidence for increased reverse Na/Ca exchange activity. In previous studies, an increase in both forward and reverse Na/Ca exchange activity was also observed in cardiomyocytes overexpressing the Na/Ca exchanger (35–37).

The main interest of the present work was to show the functional consequences of Na/Ca exchanger overexpression and to determine whether the level of Na/Ca exchange activity may shape stimulus-induced cytosolic Ca²⁺ oscillations. First, Na/Ca exchanger overexpression did not affect basal [Ca²⁺]_i, a finding compatible with the view that Na/Ca exchange displays a low affinity but high capacity for Ca²⁺ and that it does not participate in the fine tuning of [Ca²⁺]_i around the basal level (5). In no other studies of Na/Ca overexpression could a change in basal [Ca²⁺]_i be observed (35–37). Second, Na/Ca overexpression increased Ca²⁺ entry and the rise in [Ca²⁺]_i in response to membrane depolarization (Fig. 4 and 5). Likewise, it accelerated the decrease in [Ca²⁺]_i due to membrane repolarization (Fig. 5). This result indicates that within the range of the changes in [Ca²⁺]_i provoked by K⁺ (150–1,000 nmol/l), Na/Ca exchange plays a significant role in the rat β-cell [Ca²⁺]_i homeostasis, with the exchanger being a versatile system allowing both Ca²⁺ entry and outflow. In agreement with such a view, Na/Ca exchanger overexpression was associated with an increase in ⁴⁵Ca uptake whether at low or high glucose concentrations. Because this increase was seen at both glucose concentrations, it probably results from a higher ⁴⁵Ca²⁺ turnover rate leading to a faster access to radioisotopic equilibrium. It constitutes a further proof for an increased forward and reverse Na/Ca exchange activity in our transfected cells and indicates that glucose activates these two modes. Indeed, the difference in ⁴⁵Ca uptake between control and overexpressing cells was more marked at the high concentration than at the low concentration of glucose.

The present data are complementary to those obtained in rat β-cells treated with antisense oligonucleotides to knock down the expression of the exchanger. In the latter study, we observed a modest but significant reduction in the rise in [Ca²⁺]_i induced by membrane depolarization together with a major slowdown (70%) of the Ca²⁺ removal rate from the cytoplasm upon membrane repolarization (13). Taken together, these data constitute a strong indication for the prominent role played by Na/Ca exchange in its two modes of function in [Ca²⁺]_i homeostasis in rat pancreatic β-cells.

The aim of the present work was to examine the extent to which an increase in Na/Ca exchange activity could shape the electrical activity of the pancreatic β-cell and explain the differences in glucose-induced electrical activity and [Ca²⁺]_i oscillations in mouse and rat β-cells. Indeed, we previously suggested that the inward current generated by the exchange prolongs the duration of the burst of electrical activity induced by glucose, namely transforms an oscillatory pattern into continuous electrical activity. As expected, overexpression of the Na/Ca exchanger suppressed the large oscillations induced by glucose and replaced them with a staircase increase in [Ca²⁺]_i onto which oscillations of small amplitude were superimposed. Such a change was observed also when tolbutamide was used to stimulate the cells. Therefore, the difference in Na/Ca exchanger level of expression and activity (22) between the mouse and the rat β-cell may indeed explain the difference in electrical and [Ca²⁺]_i behavior between the β-cells of these two species. Such a view was confirmed by the demonstration that a slight hyperpolarization of the plasma membrane induced by a low concentration of diazoxide restored large amplitude [Ca²⁺]_i oscillations in NCX1.7-overexpressing cells.

Incidentally, Na/Ca exchanger knockout using antisense oligonucleotides also led to a decrease in the Ca²⁺ oscillations induced by glucose and tolbutamide (13). However, when considering the present data and those obtained with antisense oligonucleotides, it must be kept in mind that the Na/Ca exchanger is a complex system that allows both Ca²⁺ entry and outflow and generates in addition an inward current. In comparison, the plasma membrane Ca²⁺-ATPase only extrudes Ca²⁺. Hence, with Na/Ca exchange, one may not necessarily expect opposite effects on repressing and overexpressing the exchanger. For instance, in the present study, overexpression of the exchanger influences the oscillations induced by glucose and tolbutamide mainly by generating an inward current that prolongs the duration of the burst of electrical activity, namely transforming an oscillatory pattern into a continuous electrical activity, with parallel changes in [Ca²⁺]_i. When Na/Ca exchanger is repressed by antisense oligonucleotides, the oscillations may persist (no Na/Ca exchange inward current) but could be altered by a reduction in Ca²⁺ inflow and outflow.

It has been shown that the ability of mouse β-cells to display large [Ca²⁺]_i oscillations is closely related to their handling of Na⁺ (29). For instance, amino acids cotransported with Na⁺ have been found to transform the slow [Ca²⁺]_i oscillations induced by glucose into a sustained increase. Likewise, inhibition of the Na⁺/K⁺ pump by the removal of extracellular K⁺ or addition of ouabain leads to a similar transformation (29). Therefore, the β-cell of the rat, by having a higher rate of Na/Ca exchange activity than that of the mouse, differs from the latter in a similar respect (Na⁺ handling), explaining the differences in glucose-induced electrical activity and [Ca²⁺]_i oscillations between these cells. Thus, a higher rate of Na/Ca exchange would lead to a higher rate of Na⁺ entry and increase in cytosolic Na⁺ concentration similar to that obtained with ouabain and amino acids cotransported with Na⁺. Furthermore, the rapidly descending phases of mouse β-cell [Ca²⁺]_i oscillations have been proposed to result from the outward transport of Ca²⁺_i from the cell, a finding compatible with the view that glucose stimulates such an outward transport (29). Consequently, it can be expected that enhanced removal of Ca²⁺ from the cytoplasm would prevent the establishment of the critical [Ca²⁺]_i required for a feedback inhibition of a glucose-stimulated entry of the ion (29). Such a view is consistent with the observation that the acceleration of the outward transport of Ca²⁺ by protein kinase activation suppresses the slow [Ca²⁺]_i oscillations seen in mice (29). Such a mechanism might explain why the β-cell of the rat, which shows a higher rate of forward Na/Ca exchange activity than that of the mouse and hence a higher rate of Ca²⁺ extrusion, display less slow [Ca²⁺]_i oscillations than the β-cell of the mouse (13). Human β-cells also display [Ca²⁺]_i oscillations in response to glucose (29), which, however, appear to be closer to those of rat β-cells than to those of mouse β-cells. It would be of interest to measure the activity of the exchanger in such human β-cells.

In conclusion, the present data confirm that Na/Ca exchange plays a significant role in the rat β-cell [Ca²⁺]_i homeostasis and show that the current generated by the exchanger appears to shape stimulus-induced membrane potential and [Ca²⁺]_i oscillations in insulin-secreting cells, with the difference in electrical activity and [Ca²⁺]_i behavior seen in mouse and rat β-cells resulting in part from a difference in Na/Ca exchange activity between these two cells.

This work was supported by the Belgian Fund for Scientific Research (FRSM 3.4562.00), of which F.V.E. is a Post Doctoral Researcher.

The authors thank R. Kiss (Laboratoire d’Histopathologie) for help in immunofluorescence microscopy and C. Pastiels and A. Van Praet for technical help.