Identification and subcellular localization of the Na+/H+ exchanger and a novel related protein in the endocrine pancreas and adrenal medulla

in Journal of Molecular Endocrinology
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  • 1 1Units of Pathology,
  • 2 1Nephrology,
  • 3 2Endocrinology and Metabolism, Faculty of Medicine, Université Catholique de Louvain, Avenue Hippocrate, B-1200 Brussels, Belgium

Na+/H+ exchangers (NHE) constitute a family of membrane antiporters that contribute to the regulation of cellular pH and volume in many tissues, including pancreatic islets. We investigated the molecular identity of NHE in rodent and human endocrine pancreas, and determined its cellular and subcellular localization. NHE1 was the most abundantly expressed isoform in rat islets, and was also expressed in mouse and human islets. By western blot, an antiserum raised against the C-terminus end of NHE1 confirmed the presence of a ~100 kDa protein corresponding to NHE1 in islets and unexpectedly unveiled the existence of a ~65 kDa cross-reactive NHE1-related protein. By immunohistochemistry, the antiserum labelled the membranes of pancreatic acini and ducts, but also diffusely stained the cytoplasm of insulin, glucagon and somatostatin cells as well as endocrine cells of the adrenal medulla. Electron microscopy localized the NHE1 immunoreactivity in the membrane of secretory granules, an unexpected finding supported by a decrease in immunohistochemical signal in degranulated β-cells. Islets of Slc9A1swe/swe mice, which lack full NHE1 protein, were found to express an mRNA corresponding to the 3′ end of NHE1 as well as the ~65 kDa protein. They still showed the cytoplasmic labelling but no plasma membrane was stained. We conclude that both the full-length and the shorter-splice variant of NHE1 are expressed in all cell types of the endocrine pancreas and in the adrenal medulla of rodents and humans. The complete protein is addressed to the plasma membrane and the shorter one to the membrane of secretory granules where its function remains to be established.

Abstract

Na+/H+ exchangers (NHE) constitute a family of membrane antiporters that contribute to the regulation of cellular pH and volume in many tissues, including pancreatic islets. We investigated the molecular identity of NHE in rodent and human endocrine pancreas, and determined its cellular and subcellular localization. NHE1 was the most abundantly expressed isoform in rat islets, and was also expressed in mouse and human islets. By western blot, an antiserum raised against the C-terminus end of NHE1 confirmed the presence of a ~100 kDa protein corresponding to NHE1 in islets and unexpectedly unveiled the existence of a ~65 kDa cross-reactive NHE1-related protein. By immunohistochemistry, the antiserum labelled the membranes of pancreatic acini and ducts, but also diffusely stained the cytoplasm of insulin, glucagon and somatostatin cells as well as endocrine cells of the adrenal medulla. Electron microscopy localized the NHE1 immunoreactivity in the membrane of secretory granules, an unexpected finding supported by a decrease in immunohistochemical signal in degranulated β-cells. Islets of Slc9A1swe/swe mice, which lack full NHE1 protein, were found to express an mRNA corresponding to the 3′ end of NHE1 as well as the ~65 kDa protein. They still showed the cytoplasmic labelling but no plasma membrane was stained. We conclude that both the full-length and the shorter-splice variant of NHE1 are expressed in all cell types of the endocrine pancreas and in the adrenal medulla of rodents and humans. The complete protein is addressed to the plasma membrane and the shorter one to the membrane of secretory granules where its function remains to be established.

Introduction

Pancreatic β cells adjust insulin secretion to the ambient concentration of glucose and other nutrients through changes in their metabolism (Newgard 2002, MacDonald et al. 2005a, Matschinsky et al. 2006). Oxidative glycolysis increases the ATP:ADP ratio, which closes ATP-sensitive K+ channels in the plasma membrane, thereby causing Ca2+ influx through voltage-dependent Ca2+ channels and rise in the concentration of cytosolic Ca2+ (Seino et al. 2000, Gilon et al. 2002, MacDonald et al. 2005b). This rise triggers exocytosis of insulin-containing granules. Simultaneously, but independently of its action on ATP-sensitive K+ channels, the metabolism of glucose produces amplifying signals that augment secretion without further increasing Ca2+ (Henquin 2000, Aizawa et al. 2002, Straub & Sharp 2002). During stimulation by nutrients, the β cell cytosolic pH (pHi; Lindstrom & Sehlin 1984, Best et al. 1988, Juntti-Berggren et al. 1991, Shepherd & Henquin 1995, Salgado et al. 1996, Shepherd et al. 1996) and volume (Miley et al. 1997) increase. The functional significance of these changes is still debated (Pace et al. 1983, Lindstrom & Sehlin 1986, Bertrand et al. 2002, Gunawardana & Sharp 2002) and the underlying mechanisms are incompletely elucidated. However, experiments using ionic substitutions in the extracellular medium or pharmacological tools (e.g. dimethyl-amiloride to block Na+/H+countertransport) have established that, besides HCO3/Cl exchangers, a Na+/H+ exchanger is implicated in the regulation of β cell pHi (Juntti-Berggren et al. 1991, Shepherd & Henquin 1995, Shepherd et al. 1996) and possibly volume (Miley et al. 1998). Similarly, Na+/H+exchange has been implicated in the control of pHi and volume of adrenal chromaffin cells (Delpire et al. 1988, Kao et al. 1991).

Sodium–proton exchangers (NHE) are widely distributed integral membrane proteins that regulate cellular volume and pH (Orlowski & Grinstein 1997, Ritter et al. 2001). The SLC9 family comprises many pseudogenes and genes that encode at least nine isoforms of the NHE proteins (Orlowski & Grinstein 2004, Nakamura et al. 2005). Several SLC9 genes are also known to give rise to multiple transcripts or partial mRNA (Orlowski & Grinstein 2004). The first five isoforms (NHE1 to NHE5) are well characterized and display distinct physiological and pharmacological properties. NHE1 is ubiquitous. In polarized cells, it is usually inserted in the basolateral domain of the plasma membrane where it fulfils housekeeping regulation of cell volume and pH (Orlowski & Grinstein 2004). NHE2 and NHE3 are mainly found at the apical pole of epithelial cells in kidney (Chambrey et al. 1998), intestine (Chu et al. 2002) and duct cells of salivary glands and pancreas (Lee et al. 1998, 2000), where they play a role in Na+ and fluid absorption, and secretion of protons (Orlowski & Grinstein 2004). NHE4 has been identified in the macula densa of the kidney (Peti-Peterdi et al. 2000) and in the stomach (Rossmann et al. 2001). NHE5 has mainly been found in the brain where it seems to behave like NHE3 (Baird et al. 1999). Although NHE1 to NHE5 are usually localized in the plasma membrane, NHE3 and NHE5 have also been observed in recycling vesicles (Kurashima et al. 1998, Szaszi et al. 2002). In contrast, the ubiquitously distributed NHE6 to NHE9 have not been localized to the plasma membrane but to intracellular organelles (endosomes, trans-Golgi network or mitochondria; Nakamura et al. 2005).

The primary aims of the present study were to determine the molecular identity of NHE in human and rodent islets and to establish its subcellular localization. The initial results unexpectedly led to the discovery of a novel protein related to NHE1 and heavily concentrated in the membrane of secretory granules in all cell types of the endocrine pancreas, and also in the adrenal medulla.

Materials and methods

Samples

All studies were approved by and conducted in accordance with the regulations of our institutional ethics committees. Human pancreases were obtained either at autopsy of normoglycaemic patients (n = 4) performed within 6 h after death, or from organ donors (n = 6). None of these patients had a disease affecting the pancreas. Autopsy samples from human kidney and heart were used as control tissue for RT-PCR. Human islets isolated (Dufrane et al. 2005) from the pancreas of one organ donor were provided by Dr D Dufrane (Experimental Surgery, Université Catholique de Louvain, Brussels, Belgium).

Normal male Wistar rats and National marine research institute (NMRI) mice were from a local colony. To evaluate NHE1 distribution after massive insulin secretion, Wistar rats received two i.p. injections of glibenclamide (5.0 mg/kg body weight) or saline at 12 h interval. Animals were fed ad libitum and decapitated 4 h after the second injection. C57BL/6.SJL, +/swe (slow wave epilepsy) mice (Cox et al. 1997) were purchased from the Jackson Laboratory (Bar Harbor, MI, USA). These heterozygous mice were mated and the resulting homozygous NHE1 null mutant (Slc9A1swe/swe) and wild-type (Slc9A1+/+) were studied at 5–7 weeks of age. Homozygous mutant mice exhibited a neurological phenotype including ataxia in the hind limbs, and seizures. Genotyping was performed as described (Cox et al. 1997) to confirm the phenotype. Rat islets were obtained by collagenase digestion of the pancreas (Jonas et al. 1998), and were used freshly or after 7 days of culture in RPMI 1640 medium containing 10 mM glucose and 0.5 g/100 ml BSA. A similar procedure was used to obtain mouse islets except that they were cultured for 18–42 h in RPMI 1640 medium containing 10% heat-inactivated FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin and 10 mm glucose.

Antibodies

Most experiments were performed with a rabbit serum directed against the C-terminus end of NHE1 (Goss et al. 1994), which was raised, characterized and kindly given by Dr M Donowitz (Johns Hopkins University School of Medicine). A mouse monoclonal antibody also directed against the C-terminus end of NHE (clone 4E9, MAB3140, Chemicon, Temecula, CA, USA) was used in a few, clearly identified, control experiments. Mouse monoclonal antibodies against insulin, glucagon and somatostatin were obtained from NovoBiolabs (HUI-018, GLU-001 and SOM-018 respectively; Bagsvaerd, Denmark).

Other reagents and supplies

Biotinylated secondary antibodies against mouse and rabbit Fab’ Ig fragments were obtained from Vector Laboratories (Burlingame, CA, USA) and Chemicon respectively. Streptavidin-peroxidase (SP) and Streptavidin-Texas-Red (STR) complexes were obtained from Roche-Diagnostics and Zymed (San Francisco, CA, USA). En-Vision detection system (EV) and Fluorescein isothiocyanate (FITC)-conjugated secondary antibodies were purchased from Dako (Glostrup, Denmark). Immunogold reagents including nanogold-labelled anti-rabbit Ig (NG-Ig) and silver enhancement solutions were obtained from Nanoprobes (Stony Brook, NY, USA). Electrophoresis reagents were from Bio-Rad and Amersham. Other reagents were obtained from Amresco (Solon, OH, USA), Sigma and J T Baker (Philipsburg, NJ, USA).

Tissue processing

Human or rodent samples were frozen in liquid nitrogen or fixed in 4% paraformaldehyde for 6 or 24 h.

Tissue proteins were extracted from snap-frozen samples (~500 mg) homogenized in (~10 ml/g tissue) ice-cold buffer (20 mM Imidazole, pH 7.2, 1 mM EDTA, 250 mM sucrose) containing protease inhibitors (Complete, Roche) by ten passes of P6 Ultra-Turrax (Labortechnik, Staufen, Germany) and sonicated (Branson Sonifier B12, Danbury, CT, USA). The homogenate was centrifuged at 1000 g for 15 min at 4 °C. The postnuclear supernatant was centrifuged at 80 000 g for 1 h at 4 °C to separate membrane and cytosolic fractions (Combet et al. 1999). The membrane pellet was suspended in the homogenization buffer and protein concentrations were determined using the Bradford method (Bio-Rad) with BSA as standard.

Total RNA was extracted from snap-frozen samples in Trizol according to manufacturer’s instructions (Life Biotechnologies, Gibco-BRL). RNA was reverse-transcribed in cDNA using random hexamers and the Superscript RNA reverse transcriptase (Gibco-BRL).

Immunoblot analysis

SDS-PAGE and immunoblotting were performed as described (Combet et al. 1999). The extracts were solubilized by heating at 95 °C for 3 min in sample buffer. Proteins (10–40 μg/lane) were separated by electrophoresis through 7.5% acrylamide slabs and transferred to nitrocellulose. Membranes were blocked for 30 min at room temperature in blotting buffer, followed by incubation with the primary antibody (anti-NHE1 at 1/2000). The membranes were then washed and incubated for 1 h at room temperature with anti-rabbit Fab’ peroxidase-labelled antibody (Dako). After washing, immunoblots were visualized with ECL-Plus reagent (Amersham).

Immunohistochemistry

Paraffin-embedded specimens were cut into 3 μm thick sections and processed as described elsewhere (Sempoux et al. 1998) including, when necessary, an antigen retrieval treatment. Primary antibodies were diluted in Tris (pH 7.4) supplemented with 1% BSA and applied overnight at 4 °C. All subsequent incubations lasted 1 h at room temperature. For double immunofluorescence experiments, anti-hormone and NHE1 antisera incubations were carried out sequentially. When necessary, a tyramine amplification step was added (Sempoux et al. 2003). The peroxidase activity was revealed by 3,3′-diaminobenzidine hypochloride (DAB: 50 mg/100 ml, pH 7.4; Fluka Chemie, Buchs, Switzerland) for 10 min. Antibodies and detailed conditions are described in Table 1. Specific optical density of the immunohistochemical signal was measured as described previously (Rahier et al. 1989).

Electron microscopy and immunogold labelling

After 24-h fixation in paraformaldehyde, small pancreas blocks were cryoprotected in PBS containing 15% sucrose for 48 h, before being frozen into liquid nitrogen. Forty micrometre thick cryosections were cut and incubated with NHE1 antiserum (1/50) for 45 min at 4 °C. After rinsing in PBS, NG-Ig (1/40) was applied for 1 h. After short fixation in 2.5% glutaraldehyde, the signal was amplified by a silver enhancer solution according to the manufacturer’s instructions. The sections were rinsed and embedded into Epon 812 and processed for electron microscopy (Rahier et al. 1989).

Radioactive RT-PCR analysis of rat NHE isoforms mRNA

The primers are indicated in Table 2. Radioactive PCR was performed as described previously (Jonas et al. 1999), with a thermal cycle profile consisting of a 10 min denaturing step at 95 °C followed by 30 cycles of amplification (1 min steps at 94, 60 and 72 °C each) and a final extension step of 10 min at 72 °C. TATAbox-binding protein (TBP) was used as control gene and amplified by a 24 cycles PCR (Jonas et al. 1999). The amplimers were then separated on a 6% polyacrylamide gel in Tris borate EDTA buffer, in parallel with a 100-bp DNA ladder. The gel was dried, and the amount of [α-32P]dCTP incorporated in each amplicon was quantified with a Cyclone Storage Phosphor System (Packard, Meriden, CT, USA). The ratio of specific product/control gene was then calculated for each sample.

Amplification of NHE1 species-specific cDNA and human NHE1 probe production for in situ hybridization

The primers used for human and rodent NHE isoforms are described in Table 2. PCRs were performed in a thermocycler 2400 (Applied Biosystems, Foster City, CA, USA) with a total volume of 25 μl mixture containing GeneAmp PCR buffer, 1.5 mM MgCl2, 200 μM deoxyNTP, 0.5 μM primers and 1 U Taq Gold polymerase (Applied Biosystems). The thermal cycle profile was 10 min denaturation at 94 °C followed by 35 cycles (30 s at 94 °C, 45 s at 62 °C and 1 min at 72 °C) and a final extension of 10 min at 72 °C. Abelson protooncogene or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were taken as controls.

The amplified DNA samples were electrophoresed on ethidium bromide agarose gel and quantified by GelDoc 2000 scanning device (Bio-Rad). The identity of NHE1 PCR product was confirmed by DNA sequence analysis using the dye terminator sequencing system on a Genetic analyser 3600 (Applied Biosystems). The PCR procedure was repeated with a no-reverse transcription control to exclude genomic DNA contamination and carry-over.

The probe production procedure for in situ hybridization was similar to that described previously for insulin-like growth factor-II including negative and sequence controls (Sempoux et al. 2003) except that a human NHE1 hybridization probe was produced from human heart using specific primers (Table 2).

In silico analyses

Electronic database searches for mouse Slc9A1 gene or mRNA matching sequences, structure and theoretical alternative splicing or start sites were available at http:// www.ensembl.org/Mus_musculus/geneview?gene=ENSMUSG00000028854&db=core (Stalker et al. 2004). RT-PCR primers were tested using blastn against all nucleotide databases available from the National Center for Biotechnology Information (NCBI) at http://www.ncbi.nlm.nih.gov/BLAST/ (Altschul et al. 1990). Alternate splicing sites were searched with online tool available at http://www.fruitfly.org/cgi-bin/seq_tools/splice.pl.

Results

Identification of NHE isoforms expressed in the endocrine pancreas

Different isoforms of NHE (NHE1–NHE5) were searched by semi-quantitative radioactive RT-PCR analysis of isolated rat islets. We found a major expression of NHE1 (Fig. 1A), whereas NHE2 was expressed in lower amounts. No signal was seen for NHE3 and NHE4, and only a weak signal was obtained for NHE5. Similar results were obtained with several preparations of both fresh and cultured islets. Control experiments carried out with a no-reverse transcription control did not yield any signal.

Since NHE1 was the predominant isoform in rat islets, we studied its expression in the human pancreas. RT-PCR showed expression of NHE1 mRNA in heart and kidney (Fig. 1B, lanes 1 and 2) and in extracts of total pancreas from three subjects (lanes 3–5). The PCR products obtained in pancreatic samples were sequenced and shown to correspond to human NHE1.

By western blot, a polyclonal antiserum raised against the C-terminus part of NHE1 (CtNHE1) detected a major protein band at ~100 kDa in pancreas membrane extracts (Fig. 1C) from three different subjects, with a similar pattern as in kidney extracts (positive control). The band pattern was also similar in rat and mouse total pancreas. However, in isolated human, rat or mouse islets, the ~100 kDa band was weaker and the predominant signal corresponded to a lower molecular weight (low MW, ~65 kDa). This low MW band was also visible in total pancreas extracts at least from rat and mouse, but with a low intensity (Fig. 1C). This inverse pattern is compatible with a greater abundance of the ~100 kDa protein in the exocrine pancreas (98% of the tissue) and of the ~65 kDa protein in the islets (2% of the tissue). No similar signal was found when cytosolic extracts of pancreas were incubated with the antiserum. Several minor bands were variably observed depending on the preparations, and a weak signal was detected above 75 kDa in human kidney and in human and rat pancreas and islets. They were considered non-specific either because of the lack of reproducibility or because a similar band was observed when membrane extracts were incubated with preimmune serum (Fig. 1C).

Localization of NHE1 in pancreas and adrenal gland

In histological sections of human pancreas, in situ hybridization identified a specific signal corresponding to NHE1 mRNA in islets and ducts (Fig. 2A). No signal was seen with an irrelevant probe (not shown). By immunohistochemistry, the CtNHE1 antiserum labelled islets in human pancreas. Islets were stained irrespective of the duration of tissue fixation (6 or 24 h), whereas labelling of the plasma membrane of acinar cells was only observed when a higher antibody concentration was used on shortly fixed tissues (Fig. 2B). In islets, the immunohistochemical signal was diffusely distributed over the cytoplasm with a stronger labelling in pericapillary regions (Fig. 2C). A linear staining suggestive of a membrane pattern was seen in selected areas of packed endocrine cells (Fig. 2D). The staining distinctly delineated the cell membrane in pancreatic ducts (Fig. 2E), but was fainter in exocrine acini (Fig. 2B). The specificity of immunolabelling was attested by the distinct basolateral labelling in proximal convoluted tubules in the kidney (Fig. 2F) and by the absence of staining when the preimmune rabbit serum was used as the primary antibody (not shown).

In sections of rat pancreas, the islets were considerably more immunolabelled with CtNHE1 antibody than the exocrine tissue. Double immunofluorescence for CtNHE1 (red) and insulin (green) resulted in a yellow signal in the β-cell core of the islets, while peripheral cells remained red (Fig. 3A). However, when the CtNHE1 antibody was combined with an anti-glucagon (Fig. 3B: green) or anti-somatostatin (Fig. 3C: green) antibody, peripheral cells of the islets were stained in yellow. These results indicate that NHE1 is present in β, α and δ cells. In all cell types, the staining was diffuse over the cytoplasm.

In human adrenals, the staining pattern by CtNHE1 differed strikingly between medulla and cortex (Fig. 4A). The serum diffusely stained the cytoplasm in the medulla (Fig. 4B), but only stained the plasma membranes in the cortex (Fig. 4C). This staining pattern was also observed in rat and mouse adrenals, irrespective of the duration of fixation (not shown).

Ultrastructural localization and prominence of the membrane localization after exocytosis

Since the signal obtained with CtNHE1 antiserum was predominantly cytoplasmic, the subcellular localization of the epitope was investigated by electron microscopy on rat pancreas. The immunogold signal was localized in areas rich in endocrine granules (Fig. 5A). The background was very low outside the granule area (nucleus, mitochondria and endoplasmic reticulum) so that the labelling density was at least tenfold higher over insulin granules and fivefold higher over glucagon granules than other intracellular structures (Fig. 5B). The membranes are not visible in our preparations because preservation of immunoreactivity imposed suboptimal fixation procedures and precluded the use of membrane-contrasting agents. Therefore, to assess the visual impression that gold particles are located at the periphery of endocrine granules, the distance between each gold particle and the centre of the closest granule was measured. The frequency distribution of these distances showed that most gold particles are separated from the centre of the granule by a distance corresponding to the average radius of the insulin (Fig. 5C) or glucagon (Fig. 5D) granules, as illustrated by the corresponding micrographs. Because of poor preservation of cell membranes and the relatively low abundance of CtNHE1 signal in acinar when compared with the endocrine cells, no specific labelling could be observed in exocrine cells (not shown).

Since insulin granules showed a major immunoreactivity against CtNHE1, we investigated the impact of a strong stimulation of insulin secretion on the signal distribution in β cells. We compared the pancreas of control rats with that of rats treated with high doses of glibenclamide. The immunoreactivity of insulin in islets from test animals was markedly decreased (by 56 ± 8%, s.d., Fig. 6A and B), reflecting β-cell degranulation (Rahier et al. 1989). As compared with controls, the cytoplasmic CtNHE1 staining was fainter in degranulated islets but the plasma membrane labelling was stronger so that the overall signal intensity was only slightly reduced (by 20 ± 9%, s.d.; Fig. 6C and D).

Identification of a novel NHE1-like protein

In control Slc9A1+/+mice, as in humans and control rats, the islets were diffusely stained by CtNHE1 antiserum, whereas only membrane staining was observed in acinar cells (Fig. 7A). In Slc9A1swe/swe mice that lack functional NHE1 protein, a diffuse labelling unexpectedly persisted in islets, while exocrine cells were negative (Fig. 7B). By contrast, hepatocytes showed no cytoplasmic staining and the selective labelling of their plasma membranes in Slc9A1+/+ mice (Fig. 7C) was completely absent in Slc9A1swe/swe mice (Fig. 7D). Adrenal medulla from Slc9A1+/+ and Slc9A1swe/swe mice showed a diffuse cytoplasmic staining pattern by CtNHE1 antiserum (Fig. 7E and F). In adrenal cortex from Slc9A1+/+ mice, the antiserum distinctly stained the cell membranes but not the cytoplasm (Fig. 7G). CtNHE1 did not stain the membranes from Slc9A1swe/swe mice adrenal cortex (Fig. 7H).

The persistence of a labelling in islets or adrenal medulla from Slc9A1swe/swe mice was surprising, particularly since the exocrine pancreas, the liver and the adrenal cortex were negative, as expected. Therefore, to determine whether this reflected a non-specific binding or revealed the presence of a fragment of NHE1 in Slc9A1swe/swe mice, RT-PCR was used to amplify defined fragments of NHE1 mRNA from liver, isolated islets and adrenals. The strategy was to seek out expression variations between full-length mRNA and a 3′ short-length mRNA downstream of the mutation point (Fig. 8A). A common 3′ primer was designed at the end of the epitope-coding region (P4). A short-end 5′ primer was chosen between the swe mutation point and the epitope-coding region (P1), and a full-length forward primer was chosen at the 5′-end exon 2 as described previously (Bell et al. 1999). The largest amplified segment of 1834 bp (5′ ex2 → P4) encompasses a large portion of the full-length NHE1 mRNA (Fig. 8B). It was only observed in tissues from Slc9A1+/+ animals. A smaller segment of 706 bp (P1 → P4) corresponding mostly to the 3′-end of the mRNA includes the region coding for the C-terminus epitope. It was observed in liver, islets and adrenals from Slc9A1+/+ as well as Slc9A1swe/swe mice (Fig. 8B). A RT-PCR using primers designed for genotyping confirmed the mutated or wild-type sequence of mRNA extracted from these tissues (Fig. 8B: 200 bp). These results indicate that the mRNA sequence which encodes the epitope recognized by CtNHE1 antiserum is present in both Slc9A1+/+ and Slc9A1swe/swe mice even if the latter lack full-length mRNA and NHE1 protein (Cox et al. 1997).

By western blot, isolated islets and adrenals from both Slc9A1+/+ and Slc9A1swe/swe mice showed a low MW band at ~65 kDa, which was absent in the liver. In contrast, the ~100 kDa protein observed in Slc9A1+/+ tissues was absent in Slc9A1swe/swe mice (Fig. 8C). These observations were confirmed by reprobing western blot membranes with the 4E9 monoclonal antibody directed against the same region of NHE1. In islets, the ~100 kDa band was fainter than that of ~65 kDa. In adrenals, the ~100 kDa band appeared much more abundant than that of ~65 kDa. We attribute this inverse pattern to the high proportion of cortical cells in the total adrenal protein extract. These findings show that although NHE1 is effectively absent from Slc9A1swe/swe islets and adrenals, the low MW protein is still present. This is consistent with the persistence of an immunohistochemical signal (Fig. 7B) and the presence of a 3′-end of NHE1 mRNA (Fig. 8B) in Slc9A1swe/swe islets and adrenal medulla. In contrast, no similar protein (Fig. 8C) and cytoplasmic labelling (Fig. 7D) were observed in liver cells.

Discussion

This study identified NHE1 as the major isoform of the Na+/H+ exchanger in rat pancreatic islets. Expression of NHE1 was also established by RT-PCR in isolated mouse islets and by in situ hybridization on histological sections of human islets.

At the protein level, the presence of NHE1 was documented by western blot and immunohistochemistry using a polyclonal antiserum directed against a 147 aa epitope at the C-terminus (CtNHE1; Goss et al. 1994). In the three examined species, the immunohistochemical labelling of CtNHE1 was stronger in islets than the exocrine pancreas. Whereas the abundance of CtNHE1 in the plasma membrane of duct cells (Roussa et al. 2001) was readily confirmed, detection of CtNHE1 in the membrane of exocrine acini (Roussa et al. 2001) was more difficult and required shorter fixation of the tissue and higher concentration of the antiserum. We acknowledge that our experimental conditions were selected for islet studies and were not optimal for the exocrine pancreas.

In islets, double immunofluorescence demonstrated the presence of CtNHE1 in α, β and δ cells of the islets. First, CtNHE1 labelled more cells than any single anti-hormone antibody, indicating the presence of CtNHE1 in additional islet cells. Second, true co-localization of CtNHE1 and glucagon, insulin or somatostatin was demonstrated by the superimposition of the fluorescent signals. Plasma membranes were clearly labelled, but the major staining was diffuse, though not homogeneous, over the cytoplasm. Often, the signal was most abundant at the advascular pole of islet cells, which is known to contain the highest density of secretory granules (Bonner-Weir 1988).

Using electron microscopy, we found CtNHE1 labelling at the periphery of secretory granules but not in other structures. Although the imposed fixation technique was not optimal to preserve intact membrane structures, the spatial distribution of the immunogold signal localized the epitope in the granule membrane. Further evidence for this localization of CtNHE1 was obtained by strong degranulation of β cells with glibenclamide. The efficacy of the treatment was attested by a major decrease in insulin immunolabelling, which corresponds to a decrease in the number of secretory granules (Channaoui & Rahier 1992). Decreasing the cytoplasmic CtNHE1 labelling also unveiled a strong labelling of the plasma membrane, compatible with the presence of NHE1 at this level. It is also possible that the protein was translocated from the granule to the plasma membrane during stimulation of exocytosis.

Western blot analysis of membrane extracts from the whole pancreas with CtNHE1 antiserum showed a major band at ~100 kDa (for a predicted MW of 91 kDa of NHE1) as previously observed in PS127A cultured cells (Goss et al. 1994) and rat exocrine pancreas (Anderie et al. 1998, Roussa et al. 2001). Several of the minor bands present in the gels (Fig. 1C) have also been observed by others (Anderie et al. 1998, Roussa et al. 2001). However, in extracts of isolated islets from humans, mice or rats, the NHE1 band at ~100 kDa was weak, whereas a low MW band at ~65 kDa was prominent. The same ~65 kDa band was present but faint in whole pancreas extracts. This inverse pattern reflects the dilution of the prominent low MW protein in the islets by the exocrine tissue, in which the ~100 kDa protein is more abundant. Our failure to detect the low MW band in extracts of human total pancreas, in spite of a cytoplasmic staining of islets and of the presence of a low MW band in isolated islets, may also be linked to a poorer preservation of autopsy material in comparison with animal organs.

Staining of adrenal glands from the three species with the CtNHE1 antiserum consistently yielded strikingly different results in the two parts of the organ. In the cortex, plasma membranes were distinctly labelled, whereas the medulla was diffusely stained. Moreover, the ~65 kDa protein was identified in extracts of adrenal gland as in total pancreatic extracts. The cytoplasmic diffuse labelling was thus present in islets and adrenal medulla, two tissues rich in densecore neuroendocrine secretory granules, but was absent from the exocrine pancreas, the adrenal cortex and the liver, tissues devoid of neuroendocrine secretory granules.

The unique results obtained with CtNHE1 antiserum in islets and adrenal medulla, viz the detection of a low MW protein and a predominant cytoplasmic topography, led us to study Slc9A1-mutant mice bearing a point-mutation, which introduces an aberrant stop codon within the coding sequence. Homozygous mutant mice (Slc9A1swe/swe) do not have the NHE1 protein in membranes from kidney, brain and stomach (Cox et al. 1997). We further show here that NHE1 is also absent from the membrane of pancreatic acini and ducts, hepatocytes and adrenal cortex in Slc9A1swe/swe animals. Totally unexpected, therefore, was the similar staining of the islets and adrenal medulla by the CtNHE1 antiserum in both Slc9A1swe/swe and Slc9A1+/+ mice.

The existence of splice variants of NHE1 (Dewey et al. 2001, Zerbini et al. 2003) or other NHE isoforms (Miyazaki et al. 2001), prompted us to search for variants in islets. We examined the NHE1 mRNA expression profile and confirmed that the full-length mRNA was present in liver and islets from Slc9A1+/+ but not Slc9A1swe/swe mice. In contrast, a downstream short-length mRNA was found in tissues from both Slc9A1+/+ and Slc9A1swe/swe mice. To exclude the hypothetical expression of a non-mutated mRNA in Slc9A1swe/swe mice, the mutation site was studied with primers designed for genotyping (Cox et al. 1997). No aberrant wild-type sequence was found in mRNA from mutated animals. Again, cross-matching of the primers with non-relevant gene products was ruled out by testing their sequence against electronic mouse genome databases. At the protein level, NHE1 in its complete form of ~100 kDa was only found in Slc9A1+/+ tissues. The low MW band was found in both Slc9A1+/+ and Slc9A1swe/swe islets, which is consistent with the mRNA studies, but was not detected in the liver despite the presence of the corresponding mRNA. It is possible that the protein, which is concentrated in large secretory granules of endocrine cells, is not synthesized or is rapidly degraded in hepatocytes that do not contain similar secretory granules.

A cross-reaction between the anti-serum and a known intracellular isoform of NHE (NHE6–NHE9) is unlikely. NHE6 (Numata et al. 1998) and NHE7 (Numata & Orlowski 2001) have higher predicted MW (~80 and ~76 kDa respectively) than the ~65 kDa band. Moreover, the intracellular NHE6, NHE7, NHE8 and NHE9 are present in the liver (Nakamura et al. 2005), but CtNHE1 did not stain liver cell cytoplasm. Finally, alignments of the cDNA sequence of these four isoforms did not identify regions corresponding to the C-terminus end of NHE1 (Nakamura et al. 2005).

The presence of a limited Slc9A1 mRNA sequence corresponding to the CtNHE1 epitope in both Slc9A1swe/swe and Slc9A1+/+ mice suggests that a truncated gene product comprising the C-terminus end of the original protein is constitutively produced in neuroendocrine cells. One possible explanation could be the presence of alternate transcripts of Slc9A1. Indeed, analysis of the whole Slc9A1 sequence identifies 119 donor and 195 acceptor sites for possible splicing using a probability threshold of 0.70. In addition to these in silico analyses, support for alternate splicing can be found in previous work. First, early after the cloning of NHE1, short-length transcripts have been described in rabbit myocardium (Dyck et al. 1992). Second, amongst two alternate transcripts detected in brain and lung from engineered Slc9A1 knockout mice, one was shown to have an in-phase downstream sequence that can produce an intact C-terminus peptide (Bell et al. 1999). However, this transcript should produce a protein of predicted MW at ~80 kDa, which does not correspond to the present ~65 kDa protein. A splice variant of NHE1 with an intact C-terminus sequence exists in erythrocytes and kidney, not in liver (pancreas was not examined), and could mediate Na:Li countertransport (Zerbini et al. 2003). Again, the predicted MW of the product (~80 kDa) differs from the present ~65 kDa protein. Finally, there are reports of splice variants of a unique gene that are either differentially expressed in tissues (Zhang et al. 2004) or addressed to different subcellular locations (Ozaita et al. 2002). All these observations are in keeping with our proposal that a splice variant of SLC9A1 is specifically expressed in the neuroendocrine cells of the pancreas and the adrenal, where it produces a ~65 kDa protein that is addressed to the membrane of the secretory granules.

In conclusion, the present study shows that both the full-length and a shorter-splice variant of NHE1 are expressed in pancreatic islets and adrenal medulla of rodents and humans. As in other cell types (Cavet et al. 1999), NHE1 is addressed to the plasma membrane where it serves its functions of cytosolic pH (Juntti-Berggren et al. 1991, Shepherd & Henquin 1995, Shepherd et al. 1996) and volume (Miley et al. 1998) regulation. The shorter protein is associated with neuroendocrine secretory vesicles. In β cells, insulin granule acidification by a proton-pump is important for proinsulin conversion by the pH-sensitive prohormone convertases (Orci et al. 1994). Recent studies also indicate that an acidic granular pH is important for Ca2+-induced insulin secretion (Barg et al. 2001, Stiernet et al. 2006). Whether the NHE1-like protein is involved in the regulation of granular pH is an interesting possibility to be investigated in future work.

Table 1

Immunohistochemical staining

Antibody; dilutionAmplification systemDetection system
EV, En-Vision; 2B, biotinylated secondary antibody (1/500); 2F, secondary antibody FITC conjugate (1/20); BT, biotinylated tyramine; SP, streptavidin-peroxydase complex (1/500); STR, streptavidin-Texas Red conjugate (1/50).
Antigen retrieval
YesPolyclonal rabbit anti-NHE1; 1/2000EVDAB
YesPolyclonal rabbit anti-NHE1; 1/1000EVDAB
NoMonoclonal mouse anti-insulin; 1/10002B-SPDAB
NoPolyclonal rabbit anti-NHE1; 1/502B-SP-BT-STRFluorescence
NoMonoclonal mouse anti-insulin; 1/802FFluorescence
NoMonoclonal mouse anti-glucagon; 1/802FFluorescence
NoMonoclonal mouse anti-somatostatin; 1/802FFluorescence
Table 2

Sequences of oligonucleotide primers used for PCR analysis of NHE isoforms

Size (bp)5-Sense primer-35-Antisense primer-3Reference or GenBank Accession no.
Gene
Rat NHE1298GAA CAT CCA CCC CAA GTC TGCAG TGG GTC TGA GCC TAT GCNM_012652
Rat NHE2333AAA CCA ACC CAA GTC TAG CAT TGTCGG TTT AAG CTG TTG TCC TTC CTANM_012653
Rat NHE3282AGA GCT TCA CAT CCG TCT TAT GGGAA AGT CGC TTG ATT CCC TGT ATCM85300
Rat NHE4396CAG CGT GTT TAC CCT CTT CTA TGTTTC CGC AAA TAT CTG TGG TCA AACM85301
Rat NHE5235GCG GTC AGC CTA TCG TAT CCGCC ACT GCG TAC TGT GTC AAT CNM138858
Human NHE1492ACC ACC ACT GGA AGG ACA AGATA GGC CAG TGG GTC TGA GCM81768
Mouse NHE1: 5′ ex2–P41834ACG TCT TCT TCC TCT TCC TGC TGGGC TCC TTG CTC CGA ATC ATGBell et al. 1999; NM_012652
Mouse NHE1: P1–P4706AGG ACA TCT GTG GTC ATT ATG GCGGC TCC TTG CTC CGA ATC ATGNM_012652
Slc9A1+/+: MR0975–MR0977200CCT GAC CTG GTT CAT CAA CATCA TGC CCT GCA CAA AGA CGCox et al. 1997
Slc9A1swe/swe: MR0976–MR0977200CCT GAC CTG GTT CAT CAA CTTCA TGC CCT GCA CAA AGA CGCox et al. 1997
Figure 1
Figure 1

Expression of NHE isoforms in the pancreas and control tissues. (A) Radioactive RT-PCR analysis of NHE isoforms and TBP after 30-cycles amplification. RT-, non-reverse transcript negative control; Islets, cDNA library from isolated rat islets (representative of three experiments). Kidney and brain, cDNA libraries taken as positive controls. (B) RT-PCR for NHE1 on human tissues including heart, kidney and three different pancreas extracts. (C) Western blot analysis (20 μg/lane) of human and mouse pancreas extracts. Preimmune, membrane fraction of human pancreas incubated with preimmune serum. Cytosol, cytoplasmic fraction of the same human pancreas incubated with immune serum. Human kidney, membrane extracts from human kidney cortex as positive control for the CtNHE1 antiserum. Human pancreas, membrane extracts from human pancreas incubated with the CtNHE1 antiserum (representative of three different subjects). Human islets, membrane extracts from one human islet preparation. Rat, rat pancreas and isolated islets extracts respectively. Mouse, mouse pancreas and isolated islets extracts respectively. Low MW band (approximately 65 kDa, arrow) is present with a low intensity in total pancreas extract and high intensity in islet extract.

Citation: Journal of Molecular Endocrinology 38, 3; 10.1677/jme.1.02164

Figure 2
Figure 2

(A) In situ hybridization of human pancreas for NHE1. The digestion procedure has degraded the tissue but the signal can unambiguously be localized in islets (arrow heads) and ducts (). (B–E) Immunohistochemical staining with CtNHE1 antiserum in human pancreas. After short tissue fixation (6 h) and use of the antiserum at 1/1000, acinar plasma membranes are labelled (arrow heads) but the staining still predominates in islets (B). Longer fixation (24 h) and lower serum concentration (1/2000) unmask variations in the staining intensity of islet cells, with a major labelling of the cytoplasmic area surrounding capillaries (C: asterisk). Similar results were obtained using the antiserum at 1/200 without antigen retrieval. A faint membrane-staining pattern can be observed in selected islet areas (D: arrow heads), whereas the membrane labelling is more clear in ducts (E). (F) Immunohistochemical staining with CtNHE1 antiserum in kidney cortex. A distinct basolateral membrane pattern is present in proximal convoluted tubules whereas glomeruli () are negative. Bars: A, B, F = 50 μm; C, D, E = 20 μm.

Citation: Journal of Molecular Endocrinology 38, 3; 10.1677/jme.1.02164

Figure 3
Figure 3

Double immunofluorescence for CtNHE1 (red) and islet hormones (green) in rat pancreas. The superimposition of red and green fluorescence yields a yellow signal which indicates the co-localization of CtNHE1 and the hormone. (A) Insulin: β cells in the central part of the islet display an orange to yellow signal indicating the colocalization of CtNHE1 with insulin. Peripheral cells stained in red (CtNHE1) with no yellow signal are probably non-β cells. (B) Glucagon: the central core of the islet composed of β cells is only stained in red (CtNHE1). Peripheral cells display a green to yellow fluorescence indicating the presence of CtNHE1 in glucagon cells. (C) Somatostatin: the same pattern can be observed as for glucagon. The superimposition of red and green signals is less easily found because of the stellate shape of δ cells. However, in several areas, there is a distinct colocalization of CtNHE1 and somatostatin. Cross-reactions between antibodies were excluded by comparison with fluorescent staining obtained after simple and double immunodetection. Bars: A = 50 μm; B, C, = 20 μm.

Citation: Journal of Molecular Endocrinology 38, 3; 10.1677/jme.1.02164

Figure 4
Figure 4

Immunohistochemical staining with CtNHE1 in human adrenal. (A) Low magnification view of the adrenal medulla (left) and cortex (right). (B) High magnification view of the medulla. The neuroendocrine cells are diffusely stained by the antiserum and some cell membranes can be seen. (C) High magnification view of the cortex. The membranes of endocrine cells are stained by the antiserum, but no cytoplasmic labelling is observed. Bars: A: 100 μm; B, C: 50 μm.

Citation: Journal of Molecular Endocrinology 38, 3; 10.1677/jme.1.02164

Figure 5
Figure 5

Ultrastructural localization of CtNHE1 immunohistochemical signal. (A) Portion of β cell cytoplasm (80 000×). The signal isrestricted to the peripheral zone of the insulin granules. (B) Immunogold particles distribution between endocrine granules and other sub-cellular structures. Values represent the mean number (± s.e.) of gold particles by μm2, measured on 27 micrographs of islets from 3 animals. The signal is five- to tenfold higher over endocrine granules than in the rest of the cytoplasm. Gold particles are twice more abundant around insulin than glucagon granules. (C and D) Distribution of distances between gold particles and centre of insulin (C: n = 347) and glucagon (D: n = 200) granules. Values correspond to the proportion of gold particles found at a given distance interval from the centre of the closest insulin or glucagon granule. The electron micrograph below each histogram shows two secretory granules at a magnification matching the geometric scale of the X-axis in the histogram. The origin of this axis (zero value) is projected on the centre of the granule on the left.

Citation: Journal of Molecular Endocrinology 38, 3; 10.1677/jme.1.02164

Figure 6
Figure 6

Influence of β-cell degranulation on CtNHE1 detection in rat pancreas. (A and B) The insulin content was markedly decreased in islets from glibenclamide-treated rats as compared with control animals. (C and D) CtNHE1 staining of the same islets (step sections). Islets from control rats (C) showed a dense, diffuse labelling whereas those of glibenclamide-treated rats (D) displayed a fainter cytoplasmic labelling with a strong membrane pattern. These experiments were performed on 24 h-fixed tissue, which explains why little signal was observed in acini. Bar: 50 μm.

Citation: Journal of Molecular Endocrinology 38, 3; 10.1677/jme.1.02164

Figure 7
Figure 7

CtNHE1 immunostaining in control and Slc9A1swe/swe-mutant mice. (A and B) Pancreas. Both diffuse insular and membrane acinar immunohistochemical stainings were observed in the pancreas from Slc9A1+/+ mice (A). Only the diffuse islet labelling was observed in Slc9A1swe/swe mice (B). (C and D) Liver. The membrane labelling observed in Slc9A1+/+ mice (C) was absent in Slc9A1swe/swe mice (D). (E–H) Adrenals. A diffuse cytoplasmic staining pattern was observed in medulla from both control (E) and Slc9A1swe/swe (F) mice. A distinct membrane labelling was observed in adrenal cortex of control (G), but not of Slc9A1swe/swe (H) mice. Bars: A–D: 50 μm; E–H: 25 μm.

Citation: Journal of Molecular Endocrinology 38, 3; 10.1677/jme.1.02164

Figure 8
Figure 8

(A) Graphical representation of NHE1 mRNA. Exons are represented with alternate pattern. Introns are not represented. The mutation point in Slc9A1swe/swe (AAG-TAG: STOP) and the CtNHE1 epitope-coding sequence (Ct 147aa) are indicated. Arrow heads below represent the primers used in (B). (B) RT-PCR products using the selected primers on cDNA of liver, isolated islets and adrenals from Slc9A1+/+ and Slc9A1swe/swe mice. 5′ ex2-P4 and P1-P4 correspond to the full-length and the 3′-end of NHE1 mRNA respectively. MR0975–MR0977 and MR0976–MR0977 detect wild-type and mutated sequences respectively. (C) Western blot using polyclonal or monoclonal antibodies with liver, isolated islets and adrenal extracts (40 μg/lane) from Slc9A1+/+ and Slc9A1swe/swe mice. The ~100 kDa band corresponds to NHE1. The low MW band is similar to that shown by the arrow in Fig. 1C (mouse islets).

Citation: Journal of Molecular Endocrinology 38, 3; 10.1677/jme.1.02164

P M was supported by grant FIRST 415795 from the Walloon Region of Belgium. J C J is Senior Research Associate from the Fonds National de la Recherche Scientifique, Brussels, Belgium. This work was supported by Grants (to J R, J C J and O D) from the Fonds de la Recherche Scientifique Médicale, Brussels, and grant ARC 05/10-328 from the Direction de la Recherche Scientifique de la Communauté Française de Belgique. We are grateful to Dr M Donowitz for kindly providing the CtNHE1 antiserum and to Dr D Dufrane for providing human islets. We are also indebted to E Riveira Munoz for her expertise in genetic analysis and to Ph Camby, Y Cnops, H Debaix, M Nenquin and L Wenderickx for their skilful help. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

  • Aizawa T, Sato Y & Komatsu M 2002 Importance of nonionic signals for glucose-induced biphasic insulin secretion. Diabetes 51 (Suppl 1) S96–S98.

    • Search Google Scholar
    • Export Citation
  • Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ 1990 Basic local alignment search tool. Journal of Molecular Biology 215 403–410.

  • Anderie I, Blum R, Haase W, Grinstein S & Thevenod F 1998 Expression of NHE1 and NHE4 in rat pancreatic zymogen granule membranes. Biochemical and Biophysical Research Communications 246 330–336.

    • Search Google Scholar
    • Export Citation
  • Baird NR, Orlowski J, Szabo EZ, Zaun HC, Schultheis PJ, Menon AG & Shull GE 1999 Molecular cloning, genomic organization, and functional expression of Na+/H+ exchanger isoform 5 (NHE5) from human brain. Journal of Biological Chemistry 274 4377–4382.

    • Search Google Scholar
    • Export Citation
  • Barg S, Huang P, Eliasson L, Nelson DJ, Obermuller S, Rorsman P, Thevenod F & Renstrom E 2001 Priming of insulin granules for exocytosis by granular Cl uptake and acidification. Journal of Cell Science 114 2145–2154.

    • Search Google Scholar
    • Export Citation
  • Bell SM, Schreiner CM, Schultheis PJ, Miller ML, Evans RL, Vorhees CV, Shull GE & Scott WJ 1999 Targeted disruption of the murine Nhe1 locus induces ataxia, growth retardation, and seizures. American Journal of Physiology 276 C788–C795.

    • Search Google Scholar
    • Export Citation
  • Bertrand G, Ishiyama N, Nenquin M, Ravier MA & Henquin JC 2002 The elevation of glutamate content and the amplification of insulin secretion in glucose-stimulated pancreatic islets are not causally related. Journal of Biological Chemistry 277 32883–32891.

    • Search Google Scholar
    • Export Citation
  • Best L, Yates AP, Gordon C & Tomlinson S 1988 Modulation by cytosolic pH of calcium and rubidium fluxes in rat pancreatic islets. Biochemical Pharmacology 37 4611–4615.

    • Search Google Scholar
    • Export Citation
  • Bonner-Weir S 1988 Morphological evidence for pancreatic polarity of beta-cell within islets of Langerhans. Diabetes 37 616–621.

  • Cavet ME, Akhter S, de Medina FS, Donowitz M & Tse CM 1999 Na+/H+ exchangers (NHE1-3) have similar turnover numbers but different percentages on the cell surface. American Journal of Physiology 277 C1111–C1121.

    • Search Google Scholar
    • Export Citation
  • Chambrey R, Warnock DG, Podevin RA, Bruneval P, Mandet C, Belair MF, Bariety J & Paillard M 1998 Immunolocalization of the Na+/H+ exchanger isoform NHE2 in rat kidney. American Journal of Physiology 275 F379–F386.

    • Search Google Scholar
    • Export Citation
  • Channaoui K & Rahier J 1992 Influence of the decrease of intracellular antigenic content on morphometric analysis: effect of the type and dilution of the first antibody. Histochemistry 97 389–395.

    • Search Google Scholar
    • Export Citation
  • Chu J, Chu S & Montrose MH 2002 Apical Na+/H+ exchange near the base of mouse colonic crypts. American Journal of Physiology. Cell Physiology 283 C358–C372.

    • Search Google Scholar
    • Export Citation
  • Combet S, Van Landschoot M, Moulin P, Piech A, Verbavatz JM, Goffin E, Balligand JL, Lameire N & Devuyst O 1999 Regulation of aquaporin-1 and nitric oxide synthase isoforms in a rat model of acute peritonitis. Journal of the American Society of Nephrology 10 2185–2196.

    • Search Google Scholar
    • Export Citation
  • Cox GA, Lutz CM, Yang CL, Biemesderfer D, Bronson RT, Fu A, Aronson PS, Noebels JL & Frankel WN 1997 Sodium/hydrogen exchanger gene defect in slow-wave epilepsy mutant mice. Cell 91 139–148.

    • Search Google Scholar
    • Export Citation
  • Delpire E, Duchene C, Cornet M & Gilles R 1988 Amiloride: an inhibitor of regulatory volume decrease in rat pheochromocytoma cultured cells. Pflugers Archiv 411 223–225.

    • Search Google Scholar
    • Export Citation
  • Dewey MJ, Ennis TM & Bowman LH 2001 cDNA cloning and expression of the mouse Na/H antiporter (NHE-1) and a potential splice variant. Molecular Biology Reports 28 111–117.

    • Search Google Scholar
    • Export Citation
  • Dufrane D, Goebbels RM, Guiot Y, Squifflet JP, Henquin JC & Gianello P 2005 A simple method using a polymethylpenten chamber for isolation of human pancreatic islets. Pancreas 30 e51–e59.

    • Search Google Scholar
    • Export Citation
  • Dyck JR, Lopaschuk GD & Fliegel L 1992 Identification of a small Na+/H+ exchanger-like message in the rabbit myocardium. FEBS Letters 310 255–259.

    • Search Google Scholar
    • Export Citation
  • Gilon P, Ravier MA, Jonas JC & Henquin JC 2002 Control mechanisms of the oscillations of insulin secretion in vitro and in vivo. Diabetes 51 (Suppl 1) S144–S151.

    • Search Google Scholar
    • Export Citation
  • Goss GG, Woodside M, Wakabayashi S, Pouyssegur J, Waddell T, Downey GP & Grinstein S 1994 ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter. Analysis of phosphorylation and subcellular localization. Journal of Biological Chemistry 269 8741–8748.

    • Search Google Scholar
    • Export Citation
  • Gunawardana SC & Sharp GWG 2002 Intracellular pH plays a critical role in glucose-induced time-dependent potentiation of insulin release in rat islets. Diabetes 51 105.

    • Search Google Scholar
    • Export Citation
  • Henquin JC 2000 Triggering and amplifying pathways of regulation of insulin secretion by glucose. Diabetes 49 1751–1760.

  • Jonas JC, Gilon P & Henquin JC 1998 Temporal and quantitative correlations between insulin secretion and stably elevated or oscillatory cytoplasmic Ca2+ in mouse pancreatic beta-cells. Diabetes 47 1266–1273.

    • Search Google Scholar
    • Export Citation
  • Jonas JC, Sharma A, Hasenkamp W, Ilkova H, Patane G, Laybutt R, Bonner-Weir S & Weir GC 1999 Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes. Journal of Biological Chemistry 274 14112.

    • Search Google Scholar
    • Export Citation
  • Juntti-Berggren L, Arkhammar P, Nilsson T, Rorsman P & Berggren PO 1991 Glucose-induced increase in cytoplasmic pH in pancreatic beta-cells is mediated by Na+/H+ exchange, an effect not dependent on protein kinase C. Journal of Biological Chemistry 266 23537–23541.

    • Search Google Scholar
    • Export Citation
  • Kao LS, Ho MY & Cragoe EJ Jr. 1991 Intracellular pH and catecholamine secretion from bovine adrenal chromaffin cells. Journal of Neurochemistry 57 1656–1660.

    • Search Google Scholar
    • Export Citation
  • Kurashima K, Szabo EZ, Lukacs G, Orlowski J & Grinstein S 1998 Endosomal recycling of the Na+/H+ exchanger NHE3 isoform is regulated by the phosphatidylinositol 3-kinase pathway. Journal of Biological Chemistry 273 20828–20836.

    • Search Google Scholar
    • Export Citation
  • Lee MG, Schultheis PJ, Yan M, Shull GE, Bookstein C, Chang E, Tse M, Donowitz M, Park K & Muallem S 1998 Membrane-limited expression and regulation of Na+/H+ exchanger isoforms by P2 receptors in the rat submandibular gland duct. Journal of Physiology 513 341–357.

    • Search Google Scholar
    • Export Citation
  • Lee MG, Ahn W, Choi JY, Luo X, Seo JT, Schultheis PJ, Shull GE, Kim KH & Muallem S 2000 Na+-dependent transporters mediate HCO3 salvage across the luminal membrane of the main pancreatic duct. Journal of Clinical Investigation 105 1651–1658.

    • Search Google Scholar
    • Export Citation
  • Lindstrom P & Sehlin J 1984 Effect of glucose on the intracellular pH of pancreatic islet cells. Biochemical Journal 218 887–892.

  • Lindstrom P & Sehlin J 1986 Effect of intracellular alkalinization on pancreatic islet calcium uptake and insulin secretion. Biochemical Journal 239 199–204.

    • Search Google Scholar
    • Export Citation
  • MacDonald MJ, Fahien LA, Brown LJ, Hasan NM, Buss JD & Kendrick MA 2005a Perspective: emerging evidence for signaling roles of mitochondrial anaplerotic products in insulin secretion. American Journal of Physiology Endocrinology and Metabolism 288 E1–E15.

    • Search Google Scholar
    • Export Citation
  • MacDonald PE, Joseph JW & Rorsman P 2005b Glucose-sensing mechanisms in pancreatic beta-cells. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 360 2211–2225.

    • Search Google Scholar
    • Export Citation
  • Matschinsky FM, Magnuson MA, Zelent D, Jetton TL, Doliba N, Han Y, Taub R & Grimsby J 2006 The network of glucokinase-expressing cells in glucose homeostasis and the potential of glucokinase activators for diabetes therapy. Diabetes 55 1–12.

    • Search Google Scholar
    • Export Citation
  • Miley HE, Sheader EA, Brown PD & Best L 1997 Glucose-induced swelling in rat pancreatic beta-cells. Journal of Physiology 504 191–198.

  • Miley HE, Holden D, Grint R, Best L & Brown PD 1998 Regulatory volume increase in rat pancreatic beta-cells. Pflugers Archiv 435 227–230.

    • Search Google Scholar
    • Export Citation
  • Miyazaki E, Sakaguchi M, Wakabayashi S, Shigekawa M & Mihara K 2001 NHE6 protein possesses a signal peptide destined for endoplasmic reticulum membrane and localizes in secretory organelles of the cell. Journal of Biological Chemistry 276 49221–49227.

    • Search Google Scholar
    • Export Citation
  • Nakamura N, Tanaka S, Teko Y, Mitsui K & Kanazawa H 2005 Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation. Journal of Biological Chemistry 280 1561–1572.

    • Search Google Scholar
    • Export Citation
  • Newgard CB 2002 While tinkering with the beta-cell…metabolic regulatory mechanisms and new therapeutic strategies. Diabetes 51 3141–3150.

    • Search Google Scholar
    • Export Citation
  • Numata M & Orlowski J 2001 Molecular cloning and characterization of a novel (Na+,K+)/H+ exchanger localized to the trans-Golgi network. Journal of Biological Chemistry 276 17387–17394.

    • Search Google Scholar
    • Export Citation
  • Numata M, Petrecca K, Lake N & Orlowski J 1998 Identification of a mitochondrial Na+/H+ exchanger. Journal of Biological Chemistry 273 6951–6959.

    • Search Google Scholar
    • Export Citation
  • Orci L, Halban P, Perrelet A, Amherdt M, Ravazzola M & Anderson RGW 1994 pH-independent and -dependent cleavage of proinsulin in the same secretory vesicle. Journal of Cell Biology 126 1149–1156.

    • Search Google Scholar
    • Export Citation
  • Orlowski J & Grinstein S 1997 Na+/H+ exchangers of mammalian cells. Journal of Biological Chemistry 272 22373–22376.

  • Orlowski J & Grinstein S 2004 Diversity of the mammalian sodium/proton exchanger SLC9 gene family. Pflugers Archiv 447 549–565.

  • Ozaita A, Martone ME, Ellisman MH & Rudy B 2002 Differential subcellular localization of the two alternatively spliced isoforms of the Kv3.1 potassium channel subunit in brain. Journal of Neurophysiology 88 394–408.

    • Search Google Scholar
    • Export Citation
  • Pace CS, Tarvin JT & Smith JS 1983 Stimulus-secretion coupling in beta-cells: modulation by pH. American Journal of Physiology 244 E3–E18.

    • Search Google Scholar
    • Export Citation
  • Peti-Peterdi J, Chambrey R, Bebok Z, Biemesderfer D, St John PL, Abrahamson DR, Warnock DG & Bell PD 2000 Macula densa Na+/H+ exchange activities mediated by apical NHE2 and basolateral NHE4 isoforms. American Journal of Physiology. Renal Physiology 278 F452–F463.

    • Search Google Scholar
    • Export Citation
  • Rahier J, Stevens M, de Menten Y & Henquin JC 1989 Determination of antigen concentration in tissue sections by immunodensitometry. Laboratory Investigations 61 357–363.

    • Search Google Scholar
    • Export Citation
  • Ritter M, Fuerst J, Woll E, Chwatal S, Gschwentner M, Lang F, Deetjen P & Paulmichl M 2001 Na+/H+ exchangers: linking osmotic dysequilibrium to modified cell function. Cellular Physiology and Biochemistry 11 1–18.

    • Search Google Scholar
    • Export Citation
  • Rossmann H, Sonnentag T, Heinzmann A, Seidler B, Bachmann O, Vieillard-Baron D, Gregor M & Seidler U 2001 Differential expression and regulation of Na+/H+ exchanger isoforms in rabbit parietal and mucous cells. American Journal of Physiology. Gastrointestinal and Liver Physiology 281 G447–G458.

    • Search Google Scholar
    • Export Citation
  • Roussa E, Alper SL & Thevenod F 2001 Immunolocalization of anion exchanger AE2, Na+/H+ exchangers NHE1 and NHE4, and vacuolar type H+-ATPase in rat pancreas. Journal of Histochemistry and Cytochemistry 49 463–474.

    • Search Google Scholar
    • Export Citation
  • Salgado A, Silva AM, Santos RM & Rosario LM 1996 Multiphasic action of glucose and alpha-ketoisocaproic acid on the cytosolic pH of pancreatic beta-cells. Evidence for an acidification pathway linked to the stimulation of Ca2+ influx. Journal of Biological Chemistry 271 8738–8746.

    • Search Google Scholar
    • Export Citation
  • Seino S, Iwanaga T, Nagashima K & Miki T 2000 Diverse roles of K(ATP) channels learned from Kir6.2 genetically engineered mice. Diabetes 49 311–318.

    • Search Google Scholar
    • Export Citation
  • Sempoux C, Guiot Y, Dubois D, Nollevaux MC, Saudubray JM, Nihoul-Fekete C & Rahier J 1998 Pancreatic B-cell proliferation in persistent hyperinsulinemic hypoglycemia of infancy: an immunohistochemical study of 18 cases. Modern Pathology 11 444–449.

    • Search Google Scholar
    • Export Citation
  • Sempoux C, Guiot Y, Dahan K, Moulin P, Stevens M, Lambot V, de Lonlay P, Fournet JC, Junien C, Jaubert F et al. 2003 The focal form of persistent hyperinsulinemic hypoglycemia of infancy: morphological and molecular studies show structural and functional differences with insulinoma. Diabetes 52 784–794.

    • Search Google Scholar
    • Export Citation
  • Shepherd RM & Henquin JC 1995 The role of metabolism, cytoplasmic Ca2+, and pH-regulating exchangers in glucose-induced rise of cytoplasmic pH in normal mouse pancreatic islets. Journal of Biological Chemistry 270 7915–7921.

    • Search Google Scholar
    • Export Citation
  • Shepherd RM, Gilon P & Henquin JC 1996 Ketoisocaproic acid and leucine increase cytoplasmic pH in mouse pancreatic B cells: role of cytoplasmic Ca2+ and pH-regulating exchangers. Endocrinology 137 677–685.

    • Search Google Scholar
    • Export Citation
  • Stalker J, Gibbins B, Meidl P, Smith J, Spooner W, Hotz HR & Cox AV 2004 The Ensembl Web site: mechanics of a genome browser. Genome Research 14 951–955.

    • Search Google Scholar
    • Export Citation
  • Stiernet P, Guiot Y, Gilon P & Henquin JC 2006 Glucose acutely decreases pH of secretory granules in mouse pancreatic islets. Mechanisms and influence on insulin secretion. Journal of Biological Chemistry 281 22142–22151.

    • Search Google Scholar
    • Export Citation
  • Straub SG & Sharp GW 2002 Glucose-stimulated signaling pathways in biphasic insulin secretion. Diabetes/Metabolism Research and Reviews 18 451–463.

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  • Szaszi K, Paulsen A, Szabo EZ, Numata M, Grinstein S & Orlowski J 2002 Clathrin-mediated endocytosis and recycling of the neuron-specific Na+/H+ exchanger NHE5 isoform. Regulation by phosphatidylinositol 3′-kinase and the actin cytoskeleton. Journal of Biological Chemistry 277 42623–42632.

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  • Zerbini G, Maestroni A, Breviario D, Mangili R & Casari G 2003 Alternative splicing of NHE-1 mediates Na–Li countertransport and associates with activity rate. Diabetes 52 1511–1518.

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  • Zhang T, Haws P & Wu Q 2004 Multiple variable first exons: a mechanism for cell- and tissue-specific gene regulation. Genome Research 14 79–89.

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    Expression of NHE isoforms in the pancreas and control tissues. (A) Radioactive RT-PCR analysis of NHE isoforms and TBP after 30-cycles amplification. RT-, non-reverse transcript negative control; Islets, cDNA library from isolated rat islets (representative of three experiments). Kidney and brain, cDNA libraries taken as positive controls. (B) RT-PCR for NHE1 on human tissues including heart, kidney and three different pancreas extracts. (C) Western blot analysis (20 μg/lane) of human and mouse pancreas extracts. Preimmune, membrane fraction of human pancreas incubated with preimmune serum. Cytosol, cytoplasmic fraction of the same human pancreas incubated with immune serum. Human kidney, membrane extracts from human kidney cortex as positive control for the CtNHE1 antiserum. Human pancreas, membrane extracts from human pancreas incubated with the CtNHE1 antiserum (representative of three different subjects). Human islets, membrane extracts from one human islet preparation. Rat, rat pancreas and isolated islets extracts respectively. Mouse, mouse pancreas and isolated islets extracts respectively. Low MW band (approximately 65 kDa, arrow) is present with a low intensity in total pancreas extract and high intensity in islet extract.

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    (A) In situ hybridization of human pancreas for NHE1. The digestion procedure has degraded the tissue but the signal can unambiguously be localized in islets (arrow heads) and ducts (). (B–E) Immunohistochemical staining with CtNHE1 antiserum in human pancreas. After short tissue fixation (6 h) and use of the antiserum at 1/1000, acinar plasma membranes are labelled (arrow heads) but the staining still predominates in islets (B). Longer fixation (24 h) and lower serum concentration (1/2000) unmask variations in the staining intensity of islet cells, with a major labelling of the cytoplasmic area surrounding capillaries (C: asterisk). Similar results were obtained using the antiserum at 1/200 without antigen retrieval. A faint membrane-staining pattern can be observed in selected islet areas (D: arrow heads), whereas the membrane labelling is more clear in ducts (E). (F) Immunohistochemical staining with CtNHE1 antiserum in kidney cortex. A distinct basolateral membrane pattern is present in proximal convoluted tubules whereas glomeruli () are negative. Bars: A, B, F = 50 μm; C, D, E = 20 μm.

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    Double immunofluorescence for CtNHE1 (red) and islet hormones (green) in rat pancreas. The superimposition of red and green fluorescence yields a yellow signal which indicates the co-localization of CtNHE1 and the hormone. (A) Insulin: β cells in the central part of the islet display an orange to yellow signal indicating the colocalization of CtNHE1 with insulin. Peripheral cells stained in red (CtNHE1) with no yellow signal are probably non-β cells. (B) Glucagon: the central core of the islet composed of β cells is only stained in red (CtNHE1). Peripheral cells display a green to yellow fluorescence indicating the presence of CtNHE1 in glucagon cells. (C) Somatostatin: the same pattern can be observed as for glucagon. The superimposition of red and green signals is less easily found because of the stellate shape of δ cells. However, in several areas, there is a distinct colocalization of CtNHE1 and somatostatin. Cross-reactions between antibodies were excluded by comparison with fluorescent staining obtained after simple and double immunodetection. Bars: A = 50 μm; B, C, = 20 μm.

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    Immunohistochemical staining with CtNHE1 in human adrenal. (A) Low magnification view of the adrenal medulla (left) and cortex (right). (B) High magnification view of the medulla. The neuroendocrine cells are diffusely stained by the antiserum and some cell membranes can be seen. (C) High magnification view of the cortex. The membranes of endocrine cells are stained by the antiserum, but no cytoplasmic labelling is observed. Bars: A: 100 μm; B, C: 50 μm.

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    Ultrastructural localization of CtNHE1 immunohistochemical signal. (A) Portion of β cell cytoplasm (80 000×). The signal isrestricted to the peripheral zone of the insulin granules. (B) Immunogold particles distribution between endocrine granules and other sub-cellular structures. Values represent the mean number (± s.e.) of gold particles by μm2, measured on 27 micrographs of islets from 3 animals. The signal is five- to tenfold higher over endocrine granules than in the rest of the cytoplasm. Gold particles are twice more abundant around insulin than glucagon granules. (C and D) Distribution of distances between gold particles and centre of insulin (C: n = 347) and glucagon (D: n = 200) granules. Values correspond to the proportion of gold particles found at a given distance interval from the centre of the closest insulin or glucagon granule. The electron micrograph below each histogram shows two secretory granules at a magnification matching the geometric scale of the X-axis in the histogram. The origin of this axis (zero value) is projected on the centre of the granule on the left.

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    Influence of β-cell degranulation on CtNHE1 detection in rat pancreas. (A and B) The insulin content was markedly decreased in islets from glibenclamide-treated rats as compared with control animals. (C and D) CtNHE1 staining of the same islets (step sections). Islets from control rats (C) showed a dense, diffuse labelling whereas those of glibenclamide-treated rats (D) displayed a fainter cytoplasmic labelling with a strong membrane pattern. These experiments were performed on 24 h-fixed tissue, which explains why little signal was observed in acini. Bar: 50 μm.

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    CtNHE1 immunostaining in control and Slc9A1swe/swe-mutant mice. (A and B) Pancreas. Both diffuse insular and membrane acinar immunohistochemical stainings were observed in the pancreas from Slc9A1+/+ mice (A). Only the diffuse islet labelling was observed in Slc9A1swe/swe mice (B). (C and D) Liver. The membrane labelling observed in Slc9A1+/+ mice (C) was absent in Slc9A1swe/swe mice (D). (E–H) Adrenals. A diffuse cytoplasmic staining pattern was observed in medulla from both control (E) and Slc9A1swe/swe (F) mice. A distinct membrane labelling was observed in adrenal cortex of control (G), but not of Slc9A1swe/swe (H) mice. Bars: A–D: 50 μm; E–H: 25 μm.

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    (A) Graphical representation of NHE1 mRNA. Exons are represented with alternate pattern. Introns are not represented. The mutation point in Slc9A1swe/swe (AAG-TAG: STOP) and the CtNHE1 epitope-coding sequence (Ct 147aa) are indicated. Arrow heads below represent the primers used in (B). (B) RT-PCR products using the selected primers on cDNA of liver, isolated islets and adrenals from Slc9A1+/+ and Slc9A1swe/swe mice. 5′ ex2-P4 and P1-P4 correspond to the full-length and the 3′-end of NHE1 mRNA respectively. MR0975–MR0977 and MR0976–MR0977 detect wild-type and mutated sequences respectively. (C) Western blot using polyclonal or monoclonal antibodies with liver, isolated islets and adrenal extracts (40 μg/lane) from Slc9A1+/+ and Slc9A1swe/swe mice. The ~100 kDa band corresponds to NHE1. The low MW band is similar to that shown by the arrow in Fig. 1C (mouse islets).

  • Aizawa T, Sato Y & Komatsu M 2002 Importance of nonionic signals for glucose-induced biphasic insulin secretion. Diabetes 51 (Suppl 1) S96–S98.

    • Search Google Scholar
    • Export Citation
  • Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ 1990 Basic local alignment search tool. Journal of Molecular Biology 215 403–410.

  • Anderie I, Blum R, Haase W, Grinstein S & Thevenod F 1998 Expression of NHE1 and NHE4 in rat pancreatic zymogen granule membranes. Biochemical and Biophysical Research Communications 246 330–336.

    • Search Google Scholar
    • Export Citation
  • Baird NR, Orlowski J, Szabo EZ, Zaun HC, Schultheis PJ, Menon AG & Shull GE 1999 Molecular cloning, genomic organization, and functional expression of Na+/H+ exchanger isoform 5 (NHE5) from human brain. Journal of Biological Chemistry 274 4377–4382.

    • Search Google Scholar
    • Export Citation
  • Barg S, Huang P, Eliasson L, Nelson DJ, Obermuller S, Rorsman P, Thevenod F & Renstrom E 2001 Priming of insulin granules for exocytosis by granular Cl uptake and acidification. Journal of Cell Science 114 2145–2154.

    • Search Google Scholar
    • Export Citation
  • Bell SM, Schreiner CM, Schultheis PJ, Miller ML, Evans RL, Vorhees CV, Shull GE & Scott WJ 1999 Targeted disruption of the murine Nhe1 locus induces ataxia, growth retardation, and seizures. American Journal of Physiology 276 C788–C795.

    • Search Google Scholar
    • Export Citation
  • Bertrand G, Ishiyama N, Nenquin M, Ravier MA & Henquin JC 2002 The elevation of glutamate content and the amplification of insulin secretion in glucose-stimulated pancreatic islets are not causally related. Journal of Biological Chemistry 277 32883–32891.

    • Search Google Scholar
    • Export Citation
  • Best L, Yates AP, Gordon C & Tomlinson S 1988 Modulation by cytosolic pH of calcium and rubidium fluxes in rat pancreatic islets. Biochemical Pharmacology 37 4611–4615.

    • Search Google Scholar
    • Export Citation
  • Bonner-Weir S 1988 Morphological evidence for pancreatic polarity of beta-cell within islets of Langerhans. Diabetes 37 616–621.

  • Cavet ME, Akhter S, de Medina FS, Donowitz M & Tse CM 1999 Na+/H+ exchangers (NHE1-3) have similar turnover numbers but different percentages on the cell surface. American Journal of Physiology 277 C1111–C1121.

    • Search Google Scholar
    • Export Citation
  • Chambrey R, Warnock DG, Podevin RA, Bruneval P, Mandet C, Belair MF, Bariety J & Paillard M 1998 Immunolocalization of the Na+/H+ exchanger isoform NHE2 in rat kidney. American Journal of Physiology 275 F379–F386.

    • Search Google Scholar
    • Export Citation
  • Channaoui K & Rahier J 1992 Influence of the decrease of intracellular antigenic content on morphometric analysis: effect of the type and dilution of the first antibody. Histochemistry 97 389–395.

    • Search Google Scholar
    • Export Citation
  • Chu J, Chu S & Montrose MH 2002 Apical Na+/H+ exchange near the base of mouse colonic crypts. American Journal of Physiology. Cell Physiology 283 C358–C372.

    • Search Google Scholar
    • Export Citation
  • Combet S, Van Landschoot M, Moulin P, Piech A, Verbavatz JM, Goffin E, Balligand JL, Lameire N & Devuyst O 1999 Regulation of aquaporin-1 and nitric oxide synthase isoforms in a rat model of acute peritonitis. Journal of the American Society of Nephrology 10 2185–2196.

    • Search Google Scholar
    • Export Citation
  • Cox GA, Lutz CM, Yang CL, Biemesderfer D, Bronson RT, Fu A, Aronson PS, Noebels JL & Frankel WN 1997 Sodium/hydrogen exchanger gene defect in slow-wave epilepsy mutant mice. Cell 91 139–148.

    • Search Google Scholar
    • Export Citation
  • Delpire E, Duchene C, Cornet M & Gilles R 1988 Amiloride: an inhibitor of regulatory volume decrease in rat pheochromocytoma cultured cells. Pflugers Archiv 411 223–225.

    • Search Google Scholar
    • Export Citation
  • Dewey MJ, Ennis TM & Bowman LH 2001 cDNA cloning and expression of the mouse Na/H antiporter (NHE-1) and a potential splice variant. Molecular Biology Reports 28 111–117.

    • Search Google Scholar
    • Export Citation
  • Dufrane D, Goebbels RM, Guiot Y, Squifflet JP, Henquin JC & Gianello P 2005 A simple method using a polymethylpenten chamber for isolation of human pancreatic islets. Pancreas 30 e51–e59.

    • Search Google Scholar
    • Export Citation
  • Dyck JR, Lopaschuk GD & Fliegel L 1992 Identification of a small Na+/H+ exchanger-like message in the rabbit myocardium. FEBS Letters 310 255–259.

    • Search Google Scholar
    • Export Citation
  • Gilon P, Ravier MA, Jonas JC & Henquin JC 2002 Control mechanisms of the oscillations of insulin secretion in vitro and in vivo. Diabetes 51 (Suppl 1) S144–S151.

    • Search Google Scholar
    • Export Citation
  • Goss GG, Woodside M, Wakabayashi S, Pouyssegur J, Waddell T, Downey GP & Grinstein S 1994 ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter. Analysis of phosphorylation and subcellular localization. Journal of Biological Chemistry 269 8741–8748.

    • Search Google Scholar
    • Export Citation
  • Gunawardana SC & Sharp GWG 2002 Intracellular pH plays a critical role in glucose-induced time-dependent potentiation of insulin release in rat islets. Diabetes 51 105.

    • Search Google Scholar
    • Export Citation
  • Henquin JC 2000 Triggering and amplifying pathways of regulation of insulin secretion by glucose. Diabetes 49 1751–1760.

  • Jonas JC, Gilon P & Henquin JC 1998 Temporal and quantitative correlations between insulin secretion and stably elevated or oscillatory cytoplasmic Ca2+ in mouse pancreatic beta-cells. Diabetes 47 1266–1273.

    • Search Google Scholar
    • Export Citation
  • Jonas JC, Sharma A, Hasenkamp W, Ilkova H, Patane G, Laybutt R, Bonner-Weir S & Weir GC 1999 Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes. Journal of Biological Chemistry 274 14112.

    • Search Google Scholar
    • Export Citation
  • Juntti-Berggren L, Arkhammar P, Nilsson T, Rorsman P & Berggren PO 1991 Glucose-induced increase in cytoplasmic pH in pancreatic beta-cells is mediated by Na+/H+ exchange, an effect not dependent on protein kinase C. Journal of Biological Chemistry 266 23537–23541.

    • Search Google Scholar
    • Export Citation
  • Kao LS, Ho MY & Cragoe EJ Jr. 1991 Intracellular pH and catecholamine secretion from bovine adrenal chromaffin cells. Journal of Neurochemistry 57 1656–1660.

    • Search Google Scholar
    • Export Citation
  • Kurashima K, Szabo EZ, Lukacs G, Orlowski J & Grinstein S 1998 Endosomal recycling of the Na+/H+ exchanger NHE3 isoform is regulated by the phosphatidylinositol 3-kinase pathway. Journal of Biological Chemistry 273 20828–20836.

    • Search Google Scholar
    • Export Citation
  • Lee MG, Schultheis PJ, Yan M, Shull GE, Bookstein C, Chang E, Tse M, Donowitz M, Park K & Muallem S 1998 Membrane-limited expression and regulation of Na+/H+ exchanger isoforms by P2 receptors in the rat submandibular gland duct. Journal of Physiology 513 341–357.

    • Search Google Scholar
    • Export Citation
  • Lee MG, Ahn W, Choi JY, Luo X, Seo JT, Schultheis PJ, Shull GE, Kim KH & Muallem S 2000 Na+-dependent transporters mediate HCO3 salvage across the luminal membrane of the main pancreatic duct. Journal of Clinical Investigation 105 1651–1658.

    • Search Google Scholar
    • Export Citation
  • Lindstrom P & Sehlin J 1984 Effect of glucose on the intracellular pH of pancreatic islet cells. Biochemical Journal 218 887–892.

  • Lindstrom P & Sehlin J 1986 Effect of intracellular alkalinization on pancreatic islet calcium uptake and insulin secretion. Biochemical Journal 239 199–204.

    • Search Google Scholar
    • Export Citation
  • MacDonald MJ, Fahien LA, Brown LJ, Hasan NM, Buss JD & Kendrick MA 2005a Perspective: emerging evidence for signaling roles of mitochondrial anaplerotic products in insulin secretion. American Journal of Physiology Endocrinology and Metabolism 288 E1–E15.

    • Search Google Scholar
    • Export Citation
  • MacDonald PE, Joseph JW & Rorsman P 2005b Glucose-sensing mechanisms in pancreatic beta-cells. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 360 2211–2225.

    • Search Google Scholar
    • Export Citation
  • Matschinsky FM, Magnuson MA, Zelent D, Jetton TL, Doliba N, Han Y, Taub R & Grimsby J 2006 The network of glucokinase-expressing cells in glucose homeostasis and the potential of glucokinase activators for diabetes therapy. Diabetes 55 1–12.

    • Search Google Scholar
    • Export Citation
  • Miley HE, Sheader EA, Brown PD & Best L 1997 Glucose-induced swelling in rat pancreatic beta-cells. Journal of Physiology 504 191–198.

  • Miley HE, Holden D, Grint R, Best L & Brown PD 1998 Regulatory volume increase in rat pancreatic beta-cells. Pflugers Archiv 435 227–230.

    • Search Google Scholar
    • Export Citation
  • Miyazaki E, Sakaguchi M, Wakabayashi S, Shigekawa M & Mihara K 2001 NHE6 protein possesses a signal peptide destined for endoplasmic reticulum membrane and localizes in secretory organelles of the cell. Journal of Biological Chemistry 276 49221–49227.

    • Search Google Scholar
    • Export Citation
  • Nakamura N, Tanaka S, Teko Y, Mitsui K & Kanazawa H 2005 Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation. Journal of Biological Chemistry 280 1561–1572.

    • Search Google Scholar
    • Export Citation
  • Newgard CB 2002 While tinkering with the beta-cell…metabolic regulatory mechanisms and new therapeutic strategies. Diabetes 51 3141–3150.

    • Search Google Scholar
    • Export Citation
  • Numata M & Orlowski J 2001 Molecular cloning and characterization of a novel (Na+,K+)/H+ exchanger localized to the trans-Golgi network. Journal of Biological Chemistry 276 17387–17394.

    • Search Google Scholar
    • Export Citation
  • Numata M, Petrecca K, Lake N & Orlowski J 1998 Identification of a mitochondrial Na+/H+ exchanger. Journal of Biological Chemistry 273 6951–6959.

    • Search Google Scholar
    • Export Citation
  • Orci L, Halban P, Perrelet A, Amherdt M, Ravazzola M & Anderson RGW 1994 pH-independent and -dependent cleavage of proinsulin in the same secretory vesicle. Journal of Cell Biology 126 1149–1156.

    • Search Google Scholar
    • Export Citation
  • Orlowski J & Grinstein S 1997 Na+/H+ exchangers of mammalian cells. Journal of Biological Chemistry 272 22373–22376.

  • Orlowski J & Grinstein S 2004 Diversity of the mammalian sodium/proton exchanger SLC9 gene family. Pflugers Archiv 447 549–565.

  • Ozaita A, Martone ME, Ellisman MH & Rudy B 2002 Differential subcellular localization of the two alternatively spliced isoforms of the Kv3.1 potassium channel subunit in brain. Journal of Neurophysiology 88 394–408.

    • Search Google Scholar
    • Export Citation
  • Pace CS, Tarvin JT & Smith JS 1983 Stimulus-secretion coupling in beta-cells: modulation by pH. American Journal of Physiology 244 E3–E18.

    • Search Google Scholar
    • Export Citation
  • Peti-Peterdi J, Chambrey R, Bebok Z, Biemesderfer D, St John PL, Abrahamson DR, Warnock DG & Bell PD 2000 Macula densa Na+/H+ exchange activities mediated by apical NHE2 and basolateral NHE4 isoforms. American Journal of Physiology. Renal Physiology 278 F452–F463.

    • Search Google Scholar
    • Export Citation
  • Rahier J, Stevens M, de Menten Y & Henquin JC 1989 Determination of antigen concentration in tissue sections by immunodensitometry. Laboratory Investigations 61 357–363.

    • Search Google Scholar
    • Export Citation
  • Ritter M, Fuerst J, Woll E, Chwatal S, Gschwentner M, Lang F, Deetjen P & Paulmichl M 2001 Na+/H+ exchangers: linking osmotic dysequilibrium to modified cell function. Cellular Physiology and Biochemistry 11 1–18.

    • Search Google Scholar
    • Export Citation
  • Rossmann H, Sonnentag T, Heinzmann A, Seidler B, Bachmann O, Vieillard-Baron D, Gregor M & Seidler U 2001 Differential expression and regulation of Na+/H+ exchanger isoforms in rabbit parietal and mucous cells. American Journal of Physiology. Gastrointestinal and Liver Physiology 281 G447–G458.

    • Search Google Scholar
    • Export Citation
  • Roussa E, Alper SL & Thevenod F 2001 Immunolocalization of anion exchanger AE2, Na+/H+ exchangers NHE1 and NHE4, and vacuolar type H+-ATPase in rat pancreas. Journal of Histochemistry and Cytochemistry 49 463–474.

    • Search Google Scholar
    • Export Citation
  • Salgado A, Silva AM, Santos RM & Rosario LM 1996 Multiphasic action of glucose and alpha-ketoisocaproic acid on the cytosolic pH of pancreatic beta-cells. Evidence for an acidification pathway linked to the stimulation of Ca2+ influx. Journal of Biological Chemistry 271 8738–8746.

    • Search Google Scholar
    • Export Citation
  • Seino S, Iwanaga T, Nagashima K & Miki T 2000 Diverse roles of K(ATP) channels learned from Kir6.2 genetically engineered mice. Diabetes 49 311–318.

    • Search Google Scholar
    • Export Citation
  • Sempoux C, Guiot Y, Dubois D, Nollevaux MC, Saudubray JM, Nihoul-Fekete C & Rahier J 1998 Pancreatic B-cell proliferation in persistent hyperinsulinemic hypoglycemia of infancy: an immunohistochemical study of 18 cases. Modern Pathology 11 444–449.

    • Search Google Scholar
    • Export Citation
  • Sempoux C, Guiot Y, Dahan K, Moulin P, Stevens M, Lambot V, de Lonlay P, Fournet JC, Junien C, Jaubert F et al. 2003 The focal form of persistent hyperinsulinemic hypoglycemia of infancy: morphological and molecular studies show structural and functional differences with insulinoma. Diabetes 52 784–794.

    • Search Google Scholar
    • Export Citation
  • Shepherd RM & Henquin JC 1995 The role of metabolism, cytoplasmic Ca2+, and pH-regulating exchangers in glucose-induced rise of cytoplasmic pH in normal mouse pancreatic islets. Journal of Biological Chemistry 270 7915–7921.

    • Search Google Scholar
    • Export Citation
  • Shepherd RM, Gilon P & Henquin JC 1996 Ketoisocaproic acid and leucine increase cytoplasmic pH in mouse pancreatic B cells: role of cytoplasmic Ca2+ and pH-regulating exchangers. Endocrinology 137 677–685.

    • Search Google Scholar
    • Export Citation
  • Stalker J, Gibbins B, Meidl P, Smith J, Spooner W, Hotz HR & Cox AV 2004 The Ensembl Web site: mechanics of a genome browser. Genome Research 14 951–955.

    • Search Google Scholar
    • Export Citation
  • Stiernet P, Guiot Y, Gilon P & Henquin JC 2006 Glucose acutely decreases pH of secretory granules in mouse pancreatic islets. Mechanisms and influence on insulin secretion. Journal of Biological Chemistry 281 22142–22151.

    • Search Google Scholar
    • Export Citation
  • Straub SG & Sharp GW 2002 Glucose-stimulated signaling pathways in biphasic insulin secretion. Diabetes/Metabolism Research and Reviews 18 451–463.

    • Search Google Scholar
    • Export Citation
  • Szaszi K, Paulsen A, Szabo EZ, Numata M, Grinstein S & Orlowski J 2002 Clathrin-mediated endocytosis and recycling of the neuron-specific Na+/H+ exchanger NHE5 isoform. Regulation by phosphatidylinositol 3′-kinase and the actin cytoskeleton. Journal of Biological Chemistry 277 42623–42632.

    • Search Google Scholar
    • Export Citation
  • Zerbini G, Maestroni A, Breviario D, Mangili R & Casari G 2003 Alternative splicing of NHE-1 mediates Na–Li countertransport and associates with activity rate. Diabetes 52 1511–1518.

    • Search Google Scholar
    • Export Citation
  • Zhang T, Haws P & Wu Q 2004 Multiple variable first exons: a mechanism for cell- and tissue-specific gene regulation. Genome Research 14 79–89.

    • Search Google Scholar
    • Export Citation