Opposite Effects of Glucose on Plasma Membrane Ca2+-ATPase and Na/Ca Exchanger Transcription, Expression, and Activity in Rat Pancreatic {beta}-Cells*

Helena Maria Ximenes {ddagger} § , Adama Kamagate {ddagger}, Françoise Van Eylen {ddagger} ||, Angelo Carpinelli § and André Herchuelz {ddagger} **

From the {ddagger}Laboratory of Pharmacology, Brussels University School of Medicine, Bât. GE, 808 route de Lennik, B-1070 Brussels, Belgium and the §Department of Human Physiology and Biophysics, Institute of Biomedical Science, University of São Paulo, São Paulo 05508.900, Brazil

Received for publication, December 4, 2002 , and in revised form, March 27, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
When stimulated by glucose the pancreatic {beta}-cell displays large oscillations of the intracellular free Ca2+concentration, resulting from intermittent Ca2+ entry from the outside and outflow from the inside, the latter process being mediated by the plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger (NCX). To understand the respective role of these two mechanisms, we studied the effect of glucose on PMCA and NCX transcription, expression, and activity in rat pancreatic islet cells. Glucose (11.1 and 22.2 mM) induced a parallel decrease in PMCA transcription, expression, and activity. In contrast the sugar induced a parallel increase in NCX transcription, expression, and activity. The effects of the sugar were mimicked by the metabolizable insulin secretagogue {alpha}-ketoisocaproate and persisted in the presence of the Ca2+-channel blocker nifedipine. The above results are compatible with the view that, when stimulated, the {beta}-cell switches from a low efficiency Ca2+-extruding mechanism, the PMCA, to a high capacity system, the Na/Ca exchanger, to better face the increase in Ca2+ inflow. These effects of glucose do not result from a direct effect of the sugar itself and are not mediated by the increase in intracellular free Ca2+ concentration induced by the sugar.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+ plays a major role in the mechanisms by which glucose stimulates insulin release from the pancreatic {beta}-cell. The sugar induces the release of the hormone by generating both triggering and amplifying signals through distinct pathways (1). According to the first pathway, the metabolism of the sugar by the {beta}-cell leads to an increase in intracellular ATP or ATP/ADP ratio, which closes the ATP-dependent K+ (KATP)1 channels. This closure depolarizes the plasma membrane, leading to the opening of voltage-sensitive Ca2+ channels, with subsequent increase in Ca2+ inflow and rise in cytosolic free Ca2+ concentration ([Ca2+]i), which triggers the process of exocytotic release of insulin (1). In addition to this action, glucose may further increase insulin release, a phenomenon that can be evidenced by blocking KATP channels or preventing the action of the sugar on it, while maintaining an elevated [Ca2+]i (1, 2). Recent data, however, suggest that glucose-induced membrane depolarization does not require inhibition of KATP channels and involves instead the activation of a volume-sensitive anion channel (3). Whatever the case, a rise in [Ca2+]i is absolutely required for the triggering and amplifying pathways activated by glucose. To ensure intracellular Ca2+ homeostasis, the entry of Ca2+ into the cell must be compensated by Ca2+ outflow, which in the {beta}-cell, is mediated by two mechanisms, the plasma membrane Ca2+ ATPase (PMCA) and the Na/Ca exchanger (NCX).

The PMCA belongs to the P-type family of transport ATPases. Four different genes corresponding to four isoforms PMCA1, PMCA2, PMCA3, and PMCA4 have been evidenced. Diversity among the ATPases is in addition generated by alternative splicing of the primary transcripts that may involve two major sites (for a review, see Refs. 46). In a previous work, we identified the PMCA transcripts expressed in the {beta}-cell and characterized them at these two alternative splicing sites (7).

The Na/Ca exchanger (NCX) is a mechanism extruding Ca2+ from the cell in exchange with Na+ without consuming any energy (8). Up to now, three mammalian isoforms of NCX have been cloned, NCX1, NCX2, and NCX3, representing the products of three distinct genes. Although NCX1 is widely distributed in various tissues, NCX2 and NCX3 seem to be restricted to brain and skeletal muscle. Several splice variants of NCX1 and NCX3 have been described, each exhibiting a specific tissue distribution (for a review see Refs. 9 and 10). Rat pancreatic islet cells, purified {beta}-cells, and RINm5F and BRINBD11 cells express two NCX1 splice variants: NCX1.3 and NCX1.7 (11).

Na/Ca exchange has a low affinity but a high capacity for Ca2+, whereas the Ca2+-ATPase has a high Ca2+ affinity but low capacity for the divalent cation (12). Therefore, it is considered that Na/Ca exchange takes care of large intracellular Ca2+ loads, whereas the Ca2+-ATPase performs the fine tuning of intracellular Ca2+ level around basal [Ca2+]i, namely 0.1–0.2 µM (12).

Using antisense oligonucleotides, we recently showed in the rat {beta}-cell, that Na/Ca exchange was responsible for up to 70% of Ca2+ removal from the cytoplasm upon membrane repolarization (13). In addition, different lines of evidence suggest that glucose stimulates rat Na/Ca exchange activity (14, 15). Previous work on the PMCA shows, on the contrary, that glucose inhibits PMCA activity (for a review, see Ref. 16). Thus, while two groups out of three found a direct inhibitory effect of glucose on enzyme activity (when added to the assay medium) (1719), a fourth group found no direct effect but showed that the activity of the ATPase was significantly inhibited when measured in islets previously incubated with glucose (20). Although found by three groups, glucose-induced inhibition of PMCA activity, whether by a direct or indirect effect, was found somewhat surprising and/or unexpected. It was suggested that inhibition of PMCA could contribute to the increase in [Ca2+]i that stimulates insulin release (17, 18, 20). However, such inhibition is consistent with the abovementioned view on the respective roles of PMCA and NCX in Ca2+ homeostasis (12), which in the case of the {beta}-cell, could be formulated as follows: when stimulated by glucose, the {beta}-cell is faced with a major increase in Ca2+ inflow and, therefore, switches from a low efficiency Ca2+ extruding mechanism, the PMCA, to a high capacity system, the Na/Ca exchanger.

To ascertain such a view, we measured the effect of glucose on PMCA and NCX1 isoforms transcription, expression, and activity in rat islet cells. The data reveal that such processes are indeed reduced by glucose in the case of the PMCA but increased by the sugar in the case of the NCX.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Islet Preparations—Pancreatic islets were isolated by the collagenase technique from the pancreas of fed albino rats (21). The islets were cultured in 1640 RPMI medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin (Invitrogen, Merelbeek, Belgium) and containing glucose, {alpha}-ketoisocaproate, tolbutamide, and nifedipine when required. After incubation, the islets were pelleted for direct RNA extraction or centrifuged and resuspended in specific buffers to study PMCA and Na/Ca exchanger (NCX) activity. Isolated cells were used for immunofluorescence studies and protein expression. The method used to isolate pancreatic islet cells has been described elsewhere (22).

Design of Polymerase Chain Reaction Primers—Primer sequences used are described in Table I. To amplify the putative splicing area of NCX1 (GenBankTM accession number X68191 [GenBank] ), the primers were designed based on rat heart cDNA sequence. For PMCA isoform 1 (PMCA 1) (accession number J03753 [GenBank] ), PMCA isoform 2 (PMCA 2) (accession number J03754 [GenBank] ), and PMCA isoform 3 (PMCA3) (accession number J05087 [GenBank] ), the primers were designed based on rat brain cDNA sequence. For PMCA isoform 4 (PMCA 4) (accession number U15408 [GenBank] ), the primers were designed based on rat testis cDNA sequence. For {beta}-actin (accession number V01217 [GenBank] ), primers were based on rat cytoplasmic sequence. All primers were synthesized by Amersham Biosciences (Belgium).


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TABLE I
Position and sequence of PCR primers used for PMCA and NCX1 mRNA amplification

 

Reverse Transcription and Polymerase Chain Reaction—Total RNA was isolated from rat pancreatic islets using the RNAnowTM method (Biogentex). RNA (2 µg) was heated 10 min at 70 °C, to denaturate the RNA, and reverse-transcribed for 50 min at 42 °C, using 200 units of Superscript II RT (Invitrogen), with 25 µg/ml oligo(dt) primer, 2.5 µg/ml random primer (Promega, Leiden, The Netherlands), and triphosphate nucleosides (0.5 mM each) (Roche Applied Science, Brussels, Belgium) in a 20-µl reaction volume as recommended by the manufacturer. RNA complementary to cDNA was removed using 2 units of Escherichia coli RNase H (Roche Applied Science) for 20 min at 37 °C. The medium was then diluted with 30 µl of 16 mM EDTA, and the reaction was terminated by heating the medium up to 70 °C for 15 min. Three microliters of single-strand cDNA were amplified by PCR in a 50-µl volume using a Pwo DNA polymerase kit (Roche Applied Science), 30 pmol of each primer, and 0.5 units of Pwo DNA polymerase. The amplification was conducted in a thermal cycler (GeneAmp PCR system 2400, PerkinElmer Life Sciences, Zaventem, Belgium) under the following conditions: initial denaturation at 94 °C for 2 min; 10 cycles of 94 °C for 30 s/60 °C for 30 s/72 °C for 45 s and 17 cycles of 94 °C for 30 s/60 °C for 30 s/72 °C for 45 s increased by 20 s at each cycle for PMCA1; 10 cycles of 94 °C for 30 s/56 °C for 30 s/72 °C for 45 s and 22 cycles of 94 °C for 30 s/56 °C for 30 s/72 °C for 45 s increased by 20 s at each cycle for PMCA2; 10 cycles of 94 °C for 30 s/56 °C for 30 s/72 °C for 45 s and 27 cycles of 94 °C for 30 s/57 °C for 30 s/72 °C for 45 s increased by 20 s at each cycle for PMCA3; 10 cycles of 94 °C for 30 s/58 °C for 30 s/72 °C for 45 s and 25 cycles of 94 °C for 30 s/58 °C for 30 s/72 °C for 45 s increased by 20 s at each cycle for PMCA4; 10 cycles of 94 °C for 30 s/63 °C for 30 s/72 °C for 45 s and 24 cycles of 94 °C for 30 s/63 °C for 30 s/72 °C for 45 s increased by 20 s at each cycle for NCX; and then 72 °C for 7 min (final extension).

Quantitative Comparison of PCR products—To determine the relative amounts of the NCX1 and PMCAs gene products, the semi-quantitative reverse-transcribed (RT-PCR) method was used (11). At the end of the amplification, a 10-µl aliquot was subjected to electrophoresis in 1% (w/v) agarose gel containing 0.5 µg/ml ethidium bromide in 1x TBE buffer. All experiments were carried out with 10 pmol of {beta}-actin primers as internal control. The cDNA bands were quantified by scanning densitometry.

Plasma Membrane Ca2+-ATPase Activity—The method to measure the PMCA activity was adapted from Gronda et al. (20). After the incubation period, the tubes containing the medium plus the islets were centrifuged for 1 min at 1700 x g (4 °C). The supernatant was discarded, and the precipitated islets were resuspended in 300 mM sucrose and 10 mM Tris-HCl (pH 7.24) at 4 °C. The islet suspension was then transferred to a microhomogenizer, washed twice at 4 °C, and homogenized in 1 ml of the same buffer.

The Ca2+-ATPase activity was assayed in a 0.5-ml reaction volume. The method consists of monitoring the release of 32P from [{gamma}-32P]ATP by islet homogenates incubated in a solution containing 50 mM Tris-HCl (pH 7.24), 0.1 mM ouabain, 1 mM [{gamma}-32P]ATP, 1 mM EGTA, and 20 µmol of thapsigargin. After 45 min of incubation at 37 °C, the tubes were transferred to an ice-cold water bath. After 1 min, 0.75 ml of 0.5% (w/v) ammonium molybdate in 5% (v/v) perchloric acid and then 0.6 ml of isobutanol were added to each tube. The mixture was stirred vigorously and then centrifuged at 1700 x g (4 °C). The radioactivity was measured in an aliquot of the organic phase by liquid scintillation and, from this value, the amount of inorganic phosphate liberated from ATP was calculated. Enzyme activity represents the difference between the activity measured in the medium described above in the presence or absence of 1 µM Ca2+. Protein concentration in the homogenates was determined using the Bio-Rad Dc Protein Assay (Bio-Rad, Nazareth, Belgium) with BSA as standard.

45Ca2+ Uptake—The method used for the measurement of 45Ca2+ uptake in isolated pancreatic islets cells has been described previously (11). In brief, the islets cells were preincubated at 37 °C during 30 min in 1 ml of a non-radioactive solution consisting in a Krebs-Ringer buffer (115 mM NaCl, 1 mM CaCl2,1mM MgCl2,10mM HEPES/NaOH, pH 7.4; gassed with ambient air), containing 2.8 mM glucose (Merck), 10 µM nifedipine (Calbiochem, La Jolla, CA), and then incubated at 37 °C for 5 min in 1 ml of the same medium containing in addition 45Ca2+ (10 µCi/ml). In some experiments, NaCl was iso-osmotically replaced by sucrose (241 mM, Merck). At the end of the incubation, the cells were separated from the incubation medium by using a combined lanthanum and oil technique (14). Na/Ca exchange was evaluated by measuring -dependent 45Cao uptake.

Indirect Immunofluorescence Microscopy—The islets cells were plated on coverslips and analyzed by indirect immunofluorescence microscopy 48 h after plating. After incubation, cells were washed with TBS (tris-buffered saline: 20 mM Tris, 137 mM NaCl, pH 7.2), fixed for 20 min in 4% formol at pH 7.4 (4 °C), and washed with TBS. The cells were permeabilized in solution containing 0.01% Triton X-100, 197 µM MgCl2, 19.5 µM 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 Inc., Burlingame, CA) in TBS for 20 min. The coverslips were overlaid with the primary antibody (SWant, rabbit anti-Ca2+-ATPase (isoform 1, 2, 3, or 4), and mouse anti-Na/Ca exchanger (isoform NCX1) diluted 1/1000 in TBS-1% bovine serum albumin (BSA) buffer for 1 h. Control cells were incubated in TBS-1% BSA buffer without primary antibody and washed three times with TBS. The cells were then treated with secondary antibody (Alexa FluorTM 594 goat anti-mouse or anti-rabbit 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 nM 4',6-diamidino-2-phenylindole solution (Molecular Probes) and washed twice with TBS. The coverslips were mounted with Vectashield® (Vector Laboratories Inc.), and the cells were observed with an Axioplan microscope (Zeiss, Germany) equipped with a HBO 100-watt or XBO 100-watt illuminator and a x100 objective, and photographed with a Dual-mode cooled charge-coupled device camera (C4880, Hamamatsu, Japan). Images were analyzed and quantified using TITN answares software (ALCATEL).

Computer-assisted Microscopy for Quantitative Immunofluorescence—To measure PMCA and NCX expression in {beta} cells, mean optical density was determined by means of a SAMBA 2005 computer-assisted microscope system (UNILOG, Grenoble, France) with a x40 magnification lens (BX50 microscope, aperture 0.65; Olympus, Tokyo, Japan). Mean optical density (MOD) denotes staining intensity. Twenty fields of between 60,000 and 120,000 µm2 each were scanned for each preparation (see Refs. 2325 for further details and standardization procedures). A negative histological control slide (from which the primary antibody was omitted) was analyzed for each sample. MOD values of the negative control samples were automatically subtracted from their corresponding positive samples.

Statistics—The results are expressed as means ± S.E. The statistical significance of differences between data was assessed by using analysis of variance followed by Tukey's post test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Quantitative Analysis by RT-PCR of PMCA1–4 and NCX1 in Islets of Langerhans—To determine the effect of glucose and {alpha}-ketoisocaproate on PMCA1–4 and NCX1 transcription, the quantitative RT-PCR method was used. Several precautions must be taken to ensure that the amount of the amplified fragment is quantitatively related to the amount of template. Indeed, after a certain number of cycles, PCR reaches a plateau, depending on different individual factors. Therefore, the number of cycles corresponding to the exponential phase of the PCR amplification was first determined. PCR amplification was carried out using two specific primers flanking the putative splicing areas of PMCA1, PMCA2, PMCA3, and PMCA4 and NCX1 cDNA and focusing on cycles 26–32, 30–40, 36–42, 34–39, and 32–37, respectively. As shown in Fig. 1, the linear part of the amplification process differed from one isoform to the other. For PMCA1, 2, 3, and 4 and NCX1, cycles 27, 32, 37, 35, and 33, respectively, were chosen for further work.



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FIG. 1.
Quantitative analysis of PMCA and NCX1 mRNA by RT-PCR amplification in islets cells. Semi-logarithmic plots of the relative RT-PCR amplification of PMCA1 during cycles 26–32 (A), PMCA2 during cycles 31–38 (B), PMCA3 during cycles 36–42 (C), PMCA4 during cycles 34–39 (D), and NCX1 during cycles 32–37 (E). The cDNA values were determined from the fluorescence of each PCR fragment by densitometry (arbitrary units). Data are means (±S.E.) of three determinations. Values for S.E. when not presented are smaller than the symbols.

 

Because the amount of RNA obtained from pancreatic islets was very limited, the total RNA was reverse-transcribed in the corresponding cDNA, which was then serially diluted. The data were expressed in proportion to the amount of {beta}-actin mRNA present in the respective preparations.

Effect of Glucose on PMCA and NCX1 mRNA—Pancreatic islets were cultured with 2.8 mM, 11.1 mM, and 22.2 mM glucose for 24 h. Total RNA was isolated, and quantitative RT-PCR was performed to quantify PMCA and NCX1 isoforms mRNAs. Insulinotropic concentrations of glucose (11.1 and 22.2 mM) induced a significant linear decrease in PMCA1 and PMCA2 mRNA but failed to affect PMCA3 and PMCA4 mRNA (Fig. 2). In contrast, glucose induced a linear increase in NCX1.3 mRNA and a trend toward an increase in NCX1.7 mRNA (Fig. 2). At 11.1 mM glucose, PMCA1 and PMCA2 mRNA were reduced by 13 and 28%, respectively, whereas NCX1.3 was increased by 52%. At 22.2 mM glucose, the figures obtained were 31, 51, and 66%, respectively. Representative gels showing the effect of glucose on PMCA2 and NCX1 transcription are shown in Fig. 3.



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FIG. 2.
PMCA and NCX1 transcription in islets cells after 24-h culture in the presence of various concentrations of glucose, using {beta}-actin as internal control. Semi-quantitative RT-PCR for PMCA1 (A), PMCA2 (B), PMCA3 (C), PMCA4 (D), and NCX1 (E). The cDNA values were determined from the fluorescence of each PCR fragment by densitometry (arbitrary units). Means ± S.E. refer to three to five determinations (*, p < 0.05).

 


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FIG. 3.
Representative gels of PMCA2 and NCX1 RT-PCR amplification after 24-h culture in the presence of various concentrations of glucose. A, PMCA2; B, NCX1. The PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. MW, 1-kb DNA ladder (sizes in bases).

 

To evaluate the time course of the decrease in transcription, the effect of glucose on PMCA2 and NCX1.3 mRNA levels were measured over shorter periods of time (3 and 6 h). Fig. 4 shows that high glucose affected PMCA2 and NCX1.3 mRNA levels after 6 h of incubation. No significant effect of glucose was observed after 3 h (data not shown).



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FIG. 4.
PMCA2 and NCX1.3 transcription in islets cells after 6-h culture in the presence of various concentrations of glucose, using {beta}-actin as internal control. Semi-quantitative RT-PCR for PMCA2 (A) and NCX1.3 (B). The cDNA values were determined from the fluorescence of each PCR fragment by densitometry (arbitrary units). Means ± S.E. refer to three determinations (*, p < 0.05).

 

Effect of Glucose on PMCA and NCX1 Expression at the Protein Level—To quantify the effect of glucose on PMCA and NCX1 expression at the protein level, quantitative immunofluorescence was used. Again, the cells were exposed to glucose 2.8 or 22.2 mM for 24 h. Exposure to the high concentration of glucose decreased PMCA2 expression by about 28% (p < 0.001) but failed to affect the expression of PMCA1, PMCA3, and PMCA4 (p > at least 0.10, Fig. 5). In contrast, the sugar increased NCX1 expression by about 28% (p < 0.001, Fig. 5). Fig. 6 shows representative images illustrating the effect of glucose on PMCA2 and NCX1 (both NCX1.3 and NCX1.7) immunofluorescence.



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FIG. 5.
PMCA and NCX1 expression at the protein level after 24-h culture in the presence of various concentrations of glucose. PMCA1 (A), PMCA2 (B), PMCA3 (C), PMCA4 (D), and NCX1 (E). Protein level was quantified by immunofluorescence. Means ± S.E. refer to three determinations (at least 20 different fields examined per sample; **, p < 0.005; ***, p < 0.0001).

 


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FIG. 6.
Representative images of immunofluorescent microscopy analysis of PMCA2 and NCX1 expression at the protein level. PMCA2 (A) and NCX1 (B) expression (red color) after 24-h culture in the presence of various concentrations of glucose, 2.8 mM (2) and 22.2 mM (3), and 10 mM {alpha}-ketoisocaproate (4) Panel 1, negative control without first antibody. The nuclei are labeled in blue (4',6-diamidino-2-phenylindole).

 

Effect of Glucose on PMCA and NCX1 Activity—Ca2+-ATPase activity was measured by monitoring the release of 32P from [{gamma}-32P]ATP by islet homogenates in the presence of ouabain and thapsigargin to ensure complete inhibition of the Na+/K+-ATPase and sarco(endoplasmic) reticulum Ca2+-ATPase activity, without affecting the PMCA. Na+/Ca2+ exchange activity was measured as intracellular Na+-dependent 45Ca2+ uptake, in the presence of 10 µM nifedipine to block the effect of glucose on voltage-sensitive Ca2+-channels. Fig. 7 shows that Ca2+-ATPase activity was decreased when cultured for 24 h in the presence of 11.1 and 22.2 mM glucose (p < 0.025), whereas Na+/Ca2+ exchange activity was increased under the same experimental conditions (p < 0.025).



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FIG. 7.
PMCA and Na/Ca exchanger activity in islets cells after 24-h culture in the presence of various concentrations of glucose. A, PMCA activity was measured as the release of 32P from [{gamma}-32P]ATP (10 µg of protein/sample). Means ± S.E. refer to four determinations (*, p < 0.05); B, Na/Ca exchange was measured as intracellular Na+-dependent 45Ca2+ uptake. Means ± S.E. refer to six determinations (*, p < 0.05).

 

Effect of {alpha}-Ketoisocaproate on PMCA and NCX1 Expression and Activity—To confirm the results obtained with glucose, another metabolized insulin secretagogue ({alpha}-ketoisocaproate) was used. Fig. 8 shows that {alpha}-ketoisocaproate (10 mM) mimicked the effect of glucose to decrease PMCA1 and PMCA2 mRNA level by about 36% (p < 0.02) and 57% (p < 0.001), respectively, while failing to affect those of PMCA3 and PMCA4. Likewise, {alpha}-ketoisocaproate increased NCX1.3 mRNA levels by about 60% (p < 0.04) and induced a trend toward an increase in NCX1.7 mRNA.



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FIG. 8.
PMCA and NCX1 transcription in islets cells after 24-h culture in the presence of 2.8 mM glucose, with or without 10 mM {alpha}-ketoisocaproate (KIC), using {beta}-actin as internal control. Semi-quantitative RT-PCR for PMCA1 (A), PMCA2 (B), PMCA3 (C), PMCA4 (D), and NCX1 (E). The cDNA values were determined from the fluorescence of each PCR fragment by densitometry (arbitrary units). Means ± S.E. refer to three to five determinations (*, p < 0.05).

 

PMCA and NCX1 expression at the protein level showed a parallel tendency (Fig. 9). Thus, {alpha}-ketoisocaproate decreased PMCA1 and PMCA2 expression by 40 and 41% (p < 0.001) while failing to affect PMCA4 expression. Surprisingly, PMCA3 expression was also reduced by {alpha}-ketoisocaproate (–32%, p < 0.001). Last, like glucose, {alpha}-ketoisocaproate increased NCX1 expression by about 20% (p < 0.01).



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FIG. 9.
PMCA and NCX1 expression at the protein level after 24-h culture in the presence of 2.8 mM glucose, with or without 10 mM {alpha}-ketoisocaproate (KIC). PMCA1 (A), PMCA2 (B), PMCA3 (C), PMCA4 (D), and NCX1 (E). Protein level was quantified by immunofluorescence. Means ± S.E. refer to three determinations (at least 20 different fields examined per sample; **, p < 0.005; ***, p < 0.0001).

 

Effect of Nifedipine on PMCA and NCX1 Expression—To gain further insight into the mechanism by with glucose modulates PMCA and NCX1 expression, the effect of the sugar was examined in the presence of the Ca2+ channel blocker nifedipine. Fig. 10 shows that nifedipine (10 µM) failed to suppress the effect of glucose on PMCA and NCX1 expression. In fact, in the presence of nifedipine, the effect of the sugar was even more marked than in its absence. Thus, in the presence of the antagonist, glucose inhibited PMCA1, 2, and 3, instead of PMCA2 alone in its absence (compare Figs. 5 and 10). Likewise, the effect of the sugar to increase NCX1 expression was of larger magnitude than in its absence, the sugar increasing NCX1 expression by 28 ± 5% and 63 ± 3% in the absence and presence of nifedipine, respectively (p < 0.0001).



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FIG. 10.
Effect of 10 µM nifedipine (Nifed) on glucose (22.2 mM)-induced changes in PMCA and NCX1 expression at the protein level after 24-h culture. PMCA1 (A), PMCA2 (B), PMCA3 (C), PMCA4 (D), and NCX1 (E). Protein level was quantified by immunofluorescence. Means ± S.E. refer to three determinations (at least 20 different fields examined per sample (**, p < 0.005; ***, p < 0.0001).

 

Ca2+ entry into the {beta}-cell through voltage-sensitive Ca2+ channels may not be the sole mechanism by which glucose rises [Ca2+]i. Other mechanisms have been identified in {beta}-cell mouse, like activation of store-operated channels and intracellular Ca2+ release through inositol-trisphosphate- or ryanodine-sensitive channels (3437). However, it is unclear to which extent such mechanisms also operate in rat {beta}-cells. Therefore, the effect of nifedipine on glucose-induced increase in [Ca2+]i was examined in rat {beta}-cells. Fig. 11 shows that, in the absence of nifedipine, glucose (11 mM) induced a rapid and oscillating increase in [Ca2+]i. Such increase was completely blocked by 10 µM nifedipine. This indicates that, in the rat {beta}-cell, mechanisms other than voltage-sensitive Ca2+ channels do not contribute significantly to glucose-induced increase in [Ca2+]i, at least in the absence of a significant increase in Ca2+ inflow through such voltage-sensitive Ca2+ channels.



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FIG. 11.
Effect of 10 µM nifedipine on glucose (11.1 mM)-induced changes in [Ca2+]i in rat {beta}-cells. A, in the absence of nifedipine. B, in the presence of 10 µM nifedipine. The bar above the curves indicates the period of exposure to glucose. The curves shown are the mean of 328 (A) and 196 traces (B) from four and three preparations, respectively.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The present work provides convincing evidence that glucose represses PMCA but increases NCX1 transcription, expression, and activity in rat pancreatic islet cells. In view of the low amounts of PMCA and NCX expressed in islet cells, expression at the protein level was examined by quantitative immunofluorescence. Previous work showed a good correlation between results obtained using Western blotting and quantitative immunohistochemistry or immunofluorescence (2628).

The data were rather homogeneous in showing a parallel decrease in mRNA and protein levels of one of the four PMCAs, PMCA2. This is of great interest because PMCA2 is expressed in a limited number of specialized tissues like neurons, at variance with PMCA1 and PMCA4, which are transcribed in most tissues and may thus represent housekeeping enzymes (2931). Furthermore, the {beta}-cell is the sole non-neuronal tissue, so far identified, that shows the presence of substantial amounts of PMCA2 at the protein level, a finding that attests the special needs of {beta}-cells in terms of Ca2+ homeostasis (7).

Glucose also decreased PMCA1 mRNA but failed to affect the PMCA1 level of expression, although the metabolized insulin secretagogue, {alpha}-ketoisocaproate, decreased both PMCA1 and PMCA2 transcription and expression. Likewise, in the presence of the Ca2+ channel blocker nifedipine, glucose reduced the expression of PMCA1, 2, and 3. Therefore, it is conceivable that, together with PMCA2, the expression of PMCA1 and PMCA3 is repressed by the sugar.

In parallel with this decrease in expression, glucose reduced global PMCA activity when measured in islets previously incubated with glucose, in agreement with Gronda et al. (20). In the latter work, these authors observed a significant decrease in Ca2+-ATPase activity during the first 3 min of exposure to glucose with subsequent return toward control values. However, the enzyme activity measured at 60 min was still below the control value, even though this difference was not significant. Our data showing a dose-related inhibition of the ATPase activity after 24 h of incubation confirm that the inhibitory of the sugar is not transient. No attempt was made in the present study to measure any direct effect of glucose on enzyme activity.

In contrast to its effects on PMCA, the sugar induced a dose-related increase in NCX1 transcription, expression, and activity. The isoform that appeared to be up-regulated at the mRNA level was NCX1.3, although there was a trend toward an increase in NCX1.7 transcription, with resulting increase in NCX1 global expression at the protein level (NCX1.3 plus NCX1.7) and activity.

To identify pancreatic {beta}-cell genes that are responsive to glucose, Webb et al. (32) recently carried out a microarray analysis of murine {beta}-cell line MIN6 cells exposed to either high (25 mM) or low (5.5 mM) glucose for 24 h. The study demonstrated that many {beta}-cell genes were glucose-responsive, with 78 demonstrating a 2.2-fold or greater change in transcript levels. The largest functional clusters identified were secretory pathway, metabolism, signaling, and transcriptional regulation transcripts. The microarray used in the latter study allowed interrogation of 6500 murine genes and expression sequence tags, including the sequences of NCX1 gene but apparently not those of the PMCA genes (32). With respect to NCX1, the study was negative but the authors only reported genes showing a 2.2-fold or greater change in transcript levels. In the present study, the maximum change did not exceed 66%. Another microarray analysis of intact human pancreatic islets using very stringent criteria also failed to show changes in PMCA and NCX1 transcripts levels (33).

The effects exerted in the present study by the metabolizable insulin secretagogue {alpha}-ketoisocaproate on PMCA and NCX1 expression and activity were similar to those induced by glucose, and even tended to be more marked than those induced by the sugar. Likewise, the effects of glucose on both PMCA and NCX1 were not suppressed by nifedipine. Although rather preliminary, the latter data suggest that the effects of glucose to modulate PMCA and NCX1 expression and activity do not result from a direct effect of the sugar or from a simple increase in Ca2+ entry or content of the {beta}-cell, but may implicate more complex mechanism such as those evoked in the amplifying pathway of the sugar. Further work is required to identify such mechanisms of regulation.

Taken as a whole, the present data confirm the view that, in response to a stimulation by glucose, the {beta}-cell switches from a low efficiency Ca2+-extruding mechanism, the PMCA, to a high capacity system, the Na/Ca exchanger, to better face the increase in Ca2+ inflow. 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 cellular preparation or tissue. The up-regulation of NCX1 by glucose may have a further advantage, because NCX1, in addition to being able to extrude Ca2+, may also contribute to Ca2+ entry through reverse Na/Ca exchange and generate an inward current that may prolong the duration of the burst of spikes of electrical activity generated by glucose and hence enhance insulin release (15). In addition, the up-regulation of NCX1 could help to protect the {beta}-cell against the deleterious actions of elevated glucose levels. Indeed, the sugar, when used at a high concentration was observed to trigger apoptosis in {beta} cells, a process that was Ca2+-dependent (38).


    FOOTNOTES
 
* This work was supported in part by The Belgian Fund for Scientific Research (Grant FRSM 3.4562.00), by the Concerted Action Islet Research European Network in the BIOMED 2 program, and by the Amerique Latine Formation Academique (ALFA) program Islet Research European and Latin American Network (IRELAN) of the European Union. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Supported by the ALFA program IRELAN of the European Union, coming from the Institute of Biomedical Science of the University of São Paulo, Brazil. Back

|| A Senior Research Assistant of The Belgian Fund for Scientific Research. Back

** To whom correspondence should be addressed. Tel.: 32-2-555-6201; Fax: 32-2-555-6370; E-mail: herchu{at}ulb.ac.be.

1 The abbreviations used are: KATP, ATP-dependent K+ channel; KIC, {alpha}-ketoisocaproate; MOD, mean optical density; NCX, Na+/Ca2+ exchanger; PMCA, plasma membrane Ca2+-ATPase; RT, reverse transcription; BSA, bovine serum albumin. Back


    ACKNOWLEDGMENTS
 
We thank R. Kiss (Laboratoire d'Histopathologie) for his help in immunofluorescence microscopy and A. Van Praet, C. Pastiels, and A. Iabkriman for technical help.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Henquin, J.-C. (2000) Diabetes 49, 1751–1760[Abstract]
  2. Aizawa, T., Komatsu, M., Asanuma, N., Sato, Y., and Sharp, G. W. (1998) Trends Pharmacol. Sci. 19, 496–499[CrossRef][Medline] [Order article via Infotrieve]
  3. Best, L. (2002) J. Membr. Biol. 185, 193–200[CrossRef][Medline] [Order article via Infotrieve]
  4. Carafoli, E. (1994) FASEB J. 8, 993–1002[Abstract/Free Full Text]
  5. Penniston, J. T., and Enyedi, A. (1994) Cell Physiol. Biochem. 4, 148–159
  6. Strehler, E. E., and Zacharias, D. A. (2001) Physiol. Rev. 81, 21–50[Abstract/Free Full Text]
  7. Kamagate, A., Herchuelz, A., Bollen, A., and Van Eylen, F. (2000) Cell Calcium 27, 231–246[CrossRef][Medline] [Order article via Infotrieve]
  8. Herchuelz, A., and Plasman, P.-O. (1992) Ann. N. Y. Acad. Sci. 639, 642–656
  9. Philipson, K. D., and Nicoll, D. A. (2000) Annu. Rev. Physiol. 62, 111–133[CrossRef][Medline] [Order article via Infotrieve]
  10. Blaustein, M. P., and Lederer, W. J. (1999) Physiol. Rev. 79, 763–854[Abstract/Free Full Text]
  11. Van Eylen, F., Svoboda, M., and Herchuelz, A. (1997) Cell Calcium 21, 185–193[CrossRef][Medline] [Order article via Infotrieve]
  12. Carafoli, E. (1988) Methods Enzymol. 157, 3–11[Medline] [Order article via Infotrieve]
  13. Van Eylen, F., Lebeau, C., Albuquerque-Silva, J., and Herchuelz, A. (1998) Diabetes 47, 1873–1880[Abstract]
  14. Plasman, P.-O., Lebrun, P., and Herchuelz, A. (1990) Am. J. Physiol. 259, E844–E850[Medline] [Order article via Infotrieve]
  15. Van Eylen, F., Diaz-Horta, O., Barez, A., Kamagate, A., Flatt, P. R., Mubagwa, K. Macianskiene, R., and Herchuelz, A. (2002) Diabetes 51, 366–375[Abstract/Free Full Text]
  16. Gagliardino, J. J., and Rossi, J. P. F. C. (1994) Diabetes Metab. Rev. 10, 1–17[Medline] [Order article via Infotrieve]
  17. Levin, S. R., Kasson, B. G., and Driessen, J. F. (1978) J. Clin. Invest. 62, 692–701[Medline] [Order article via Infotrieve]
  18. Hoenig, M., Lee, R. J., and Ferguson, D. C. (1990) Biochim. Biophys. Acta 1022, 333–338[Medline] [Order article via Infotrieve]
  19. Kotagal, N., Colca, J. R., Buscetto, D., and McDaniel, M. L. (1985) Arch. Biochem. Biophys. 238, 161–169[Medline] [Order article via Infotrieve]
  20. Gronda, C. M., Rossi, J. P., and Gagliardino, J. J. (1988) Biochim. Biophys. Acta 943, 183–189[Medline] [Order article via Infotrieve]
  21. Lacy, P. E., and Kostianovsky, M. (1967) Diabetes 16, 35–39[Medline] [Order article via Infotrieve]
  22. Gobbe, P., and Herchuelz, A. (1989) Res. Commun. Chem. Path. Pharmacol. 63, 231–249[Medline] [Order article via Infotrieve]
  23. Choufani, G., Saussez, S., Marchant, H., Bishop, P., Schüring, M. P., Danguy, A., Salmon, I., Gabius, H. J., Kiss, R., and Hassid, S. (1999) Cancer 86, 2353–2363[CrossRef][Medline] [Order article via Infotrieve]
  24. Goldschmidt, D., Decaestecker, C., Berthe, J. V., Gordower, L., Remmelink, M., Danguy, A., Pasteels, J. L., Salmon, I., and Kiss, R. (1996) Lab. Invest. 75, 295–306[Medline] [Order article via Infotrieve]
  25. Saussez, S., Marchant, H., Nagy, N., Decaestecker, C., Hassid, S., Jortay, A., Schüring, M., Gabius, H. J., Danguy, A., Salmon, I., and Kiss, R. (1998) Cancer 82, 252–260[CrossRef][Medline] [Order article via Infotrieve]
  26. Camby, I., Belot, N., Lefranc, F., Sadeghi, N., de Launoit, Y., Kaltner, H., Musette, S., Darro, F., Danguy, A., Salmon, I., Gabius, H. J., and Kiss, R. (2002) J. Neuropathol. Exp. Neurol. 617, 585–596
  27. Nagy, N., Brenner, C., Markadieu, N., Chaboteaux, C., Camby, I., Schafer, B. W., Pochet, R., Heizmann, C. W., Salmon, I., Kiss, R., and Decaestecker, C. (2001) Lab. Invest. 81, 599–612[Medline] [Order article via Infotrieve]
  28. Pilette, C., Godding, V., Kiss, R., Delos, M., Verbeken, E., Decaestecker, C., De Paepe, K., Vaerman, J. P., Decramer, M., and Sibille, Y. (2001) Am. J. Respir. Crit. Care Med. 163, 185–194[Abstract/Free Full Text]
  29. Greeb, J., and Shull, G. E. (1989) J. Biol. Chem. 264, 18569–18576[Abstract/Free Full Text]
  30. Brandt, P., Neve, R. L., Kammesheidt, A., Rhoads, R. E., and Vanaman, T. C. (1992) J. Biol. Chem. 267, 4376–4385[Abstract/Free Full Text]
  31. Stauffer, T. P., Hilfiker, H., Carafoli, E., and Strehler, E. E. (1993) J. Biol. Chem. 268, 25993–26003[Abstract/Free Full Text]
  32. Webb, G. C., Akbar, M. S., Zhao, C., and Steiner, D. F. (2000) Proc. Nat. Acad. Sci. U. S. A. 97, 5773–5778[Abstract/Free Full Text]
  33. Shalev, A., Pise-Masison, C. A., Radonovich, M., Hoffman, S. C., Hirsberg, B., Brady, J. N., and Harlan, D. M. (2002) Endocrinology 143, 3695–3698[Abstract/Free Full Text]
  34. Gilon, P., Arredouani, A., Gailly, P., Gromada, J., and Henquin, J.-C. (1999) J. Biol. Chem. 274, 20197–20205[Abstract/Free Full Text]
  35. Lemmens, R., Larsson, O., Berggren, P.-O., and Islam, S. (2001) J. Biol. Chem. 276, 9971–9977[Abstract/Free Full Text]
  36. Dyachok, O., and Gylfe, E. (2001) J. Cell Sci. 114, 2179–2186[Abstract/Free Full Text]
  37. Varadi, A., and Rutter, G. A. (2002) Diabetes 51, Suppl. 1, S190–S201[Abstract/Free Full Text]
  38. Efanova, J. B., Zaitsev, S. V., Zhivotovsky, B., Köhler, M., Efendic, S., Orrenius, S., and Berggren, P. (1998) J. Biol. Chem. 273, 33501–33507[Abstract/Free Full Text]