The elevation of glutamate content and the amplification of insulin secretion in glucose-stimulated pancreatic islets are not causally related

Glucose increases insulin secretion by raising cytoplasmic Ca(2+) ([Ca(2+)](i)) in beta-cells (triggering pathway) and augmenting the efficacy of Ca(2+) on exocytosis (amplifying pathway). It has been suggested that glutamate formed from alpha-ketoglutarate is a messenger of the amplifying pathway (Maechler, P., and Wollheim, C. B. (1999) Nature 402, 685-689). This hypothesis was tested with mouse islets depolarized with 30 mm KCl (+ diazoxide) or with a saturating concentration of sulfonylurea. Because [Ca(2+)](i) was elevated under these conditions, insulin secretion was stimulated already in 0 mm glucose. The amplification of secretion produced by glucose was accompanied by an increase in islet glutamate. However, glutamine (0.5-2 mm) markedly augmented islet glutamate without affecting insulin secretion, whereas glucose augmented secretion without influencing glutamate levels when these were elevated by glutamine. Allosteric activation of glutamate dehydrogenase by BCH (2-amino 2-norbornane carboxylic acid) lowered islet glutamate but increased insulin secretion. Similar insulin secretion thus occurred at very different cellular glutamate levels. Glutamine did not affect islet [Ca(2+)](i) and pH(i), whereas glucose and BCH slightly raised pH(i) and either slightly decreased (30 mm KCl) or increased (tolbutamide) [Ca(2+)](i). The general dissociation between changes in islet glutamate and insulin secretion refutes a role of beta-cell glutamate in the amplification of insulin secretion by glucose.     

Glucose-induced increase in cytoplasmic pH in pancreatic beta-cells is mediated by Na+/H+ exchange, an effect not dependent on protein kinase C.

Glucose-induced changes in cytoplasmic pH (pHi) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Glucose, at concentrations above 3-5 mM, depolarized the beta-cell and increased pHi, cytoplasmic free Ca2+ ([Ca2+]i), and insulin release. This increase in pHi was dependent on the presence of extracellular Na+ and was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, a blocker of Na+/H+ exchange. Stimulation of protein kinase C with phorbol ester also induced an alkalinization. However, when protein kinase C activity was down-regulated, glucose stimulation still induced alkalinization. At 20 mM glucose, 10 mM NH4Cl induced a marked rise in pHi, paralleled by repolarization, inhibition of electrical activity, and decreases in both [Ca2+]i and insulin release. Reduction in [Ca2+]i was prevented by 200 microM tolbutamide, but not by 10 mM tetraethylammonium. At 4 mM glucose, NH4Cl induced a transient increase in insulin release, without changing [Ca2+]i. Exposure of beta-cells to 10 mM sodium acetate caused a persistent decrease in pHi, an effect paralleled by a small transient increase in [Ca2+]i. Acidification per se did not change the beta-cell sensitivity to glucose, not excluding that the activity of the ATP-regulated K+ channels may be modulated by changes in pHi.     

Na+/H+ exchanger NHE3 activity and trafficking are lipid Raft-dependent

A previous study showed that approximately 25-50% of rabbit ileal brush border (BB) Na(+)/H(+) exchanger NHE3 is in lipid rafts (LR) (Li, X., Galli, T., Leu, S., Wade, J. B., Weinman E. J., Leung, G., Cheong, A., Louvard, D., and Donowitz, M. (2001) J. Physiol. (Lond.) 537, 537-552). Here, we examined the role of LR in NHE3 transport activity using a simpler system: opossum kidney (OK) cells (a renal proximal tubule epithelial cell line) containing NHE3. approximately 50% of surface (biotinylated) NHE3 in OK cells distributed in LR by density gradient centrifugation. Disruption of LR with methyl-beta-cyclodextrin (MbetaCD) decreased NHE3 activity and increased K'(H+)(i), but K(m)((Na+)) was not affected. The MbetaCD effect was completely reversed by repletion of cholesterol, but not by an inactive analog of cholesterol (cholestane-3beta,5alpha,6beta-triol). The MbetaCD effect was specific for NHE3 activity because it did not alter Na(+)-dependent l-Ala uptake. MbetaCD did not alter OK cell BB topology and did not change the surface amount of NHE3, but greatly reduced the rate of NHE3 endocytosis. The effects of inhibiting phosphatidylinositol 3-kinase and of MbetaCD on NHE3 activity were not additive, indicating a common inhibitory mechanism. In contrast, 8-bromo-cAMP and MbetaCD inhibition of NHE3 was additive, indicating different mechanisms for inhibition of NHE3 activity. Approximately 50% of BB NHE3 and only approximately 11% of intracellular NHE3 in polarized OK cells were in LR. In summary, the BB pool of NHE3 in LR is functionally active because MbetaCD treatment decreased NHE3 basal activity. The LR pool is necessary for multiple kinetic aspects of normal NHE3 activity, including V(max) and K'(H+)(i), and also for multiple aspects of NHE3 trafficking, including at least basal endocytosis and phosphatidylinositol 3-kinase-dependent basal exocytosis. Because the C-terminal domain of NHE3 is necessary for its regulation and because the changes in NHE3 kinetics with MbetaCD resemble those with second messenger regulation of NHE3, these results suggest that the NHE3 C terminus may be involved in the MbetaCD sensitivity of NHE3.     

Dimerization is crucial for the function of the Na+/H+ exchanger NHE1

The Na(+)/H(+) exchanger 1 (NHE1) exists as a homo-dimer in the plasma membranes. In the present study, we have investigated the functional significance of the dimerization, using two nonfunctional NHE1 mutants, surface-expression-deficient G309V and transport-deficient E262I. Biochemical and immunocytochemical experiments revealed that these NHE1 mutants are capable of interacting with the wild-type NHE1 and, thus, forming a heterodimer. Expression of G309V retained the wild-type NHE1 to the ER membranes, suggesting that NHE1 would first form a dimer in the ER. On the other hand, expression of E262I markedly reduced the exchange activity of the wild-type NHE1 through an acidic shift in the intracellular pH (pH(i)) dependence, suggesting that dimerization is required for exchange activity in the physiological pH(i) range. However, a dominant-negative effect of E262I was not detected when exchange activity was measured at acidic pH(i), implying that one active subunit is sufficient to catalyze ion transport when the intracellular H(+) concentration is sufficiently high. Furthermore, intermolecular cysteine cross-linking at extracellular position Ser(375) with a bifunctional sulfhydryl reagent dramatically inhibited exchange activity mainly by inducing the acidic shift of pH(i) dependence and abolished extracellular stimuli-induced activation of NHE1 without causing a large change in the affinities for extracellular Na(+) or an inhibitor EIPA. Because monofunctional sulfhydryl regents had no effect, it is likely that cross-linking inhibited the activity of NHE1 by restricting a coupled motion between the two subunits during transport. Taken together, these data support the view that dimerization of two active subunits are required for NHE1 to possess the exchange activity in the neutral pH(i) range, although each subunit is capable of catalyzing transport in the acidic pH(i) range.     

The Na+/H+‐exchanger (NHE1) generates pH nanodomains at focal adhesions

Many tumor cells are characterized by an increased net acid production. They extrude the excess protons mainly through the Na⁺/H⁺‐exchanger NHE1. An increased NHE1 activity elevates the metastatic potential of tumor cells. Cell migration, a key step in the metastatic cascade, requires the formation and release of integrin‐mediated cell–matrix contacts (focal adhesions). As NHE1 has been localized to focal adhesion sites, the present study tests the hypothesis that NHE1 generates measurable pH nanodomains right at focal adhesions. In order to ratiometrically measure pH close to the plasma membrane, we established a novel application of the total internal reflection fluorescence microscopy (TIRFM). Human melanoma cells were transfected with DsRed2‐paxillin to identify focal adhesion sites. The pH‐sensitive dyes BCECF and WGA‐fluorescein were used to measure the submembranous cytosolic and the pericellular pH, respectively. Distinct pH nanodomains were found at focal adhesions, particularly at those located at the cell front, where NHE1 was concentrated. These sites featured a remarkably alkaline cytosolic and an acidic pericellular pH and thus a much steeper proton gradient across the plasma membrane compared to the rest of the cell. The generation of pH nanodomains could be assigned to NHE1‐mediated H⁺ export because such pH domains could not be detected in NHE1‐deficient cells. Given that both integrin avidity and mechanisms contributing to adhesion turnover are pH‐sensitive, we propose that pH nanodomains at focal adhesions, locally created and maintained by NHE1 activity especially at the cell front, modulate adhesion dynamics in migrating cells. J. Cell. Physiol. 228: 1351–1358, 2013. © 2012 Wiley Periodicals, Inc.     

B-Raf associates with and activates the NHE1 isoform of the Na+/H+ exchanger

The serine/threonine kinase B-Raf is the second most frequently occurring human oncogene after Ras. Mutations of B-Raf occur with the highest incidences in melanoma, and the most common mutant, V600E, renders B-Raf constitutively active. The sodium proton exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein responsible for regulating intracellular pH, cell volume, cell migration, and proliferation. A screen of protein kinases that bind to NHE1 revealed that B-Raf bound to the cytosolic regulatory tail of NHE1. Immunoprecipitation of NHE1 from HeLa and HEK cells confirmed the association of B-Raf with NHE1 in vivo. The expressed and purified C-terminal 182 amino acids of the NHE1 protein were also shown to associate with B-Raf protein in vitro. Because treatment with the kinase inhibitor sorafenib decreased NHE1 activity in HeLa and HEK cells, we examined the role of B-Raf in regulating NHE1 in malignant melanoma cells. Melanoma cells with the B-Raf(V600E) mutation demonstrated increased resting intracellular pH that was dependent on elevated NHE1 activity. NHE1 activity after an acute acid load was also elevated in these cell lines. Moreover, inhibition of B-Raf activity by either sorafenib, PLX4720, or siRNA reduction of B-Raf levels abolished ERK phosphorylation and decreased NHE1 activity. These results demonstrate that B-Raf associates with and stimulates NHE1 activity and that B-Raf(V600E) also increases NHE1 activity that raises intracellular pH.     

Structure and function of the NHE1 isoform of the Na+/H+ exchanger.

The Na+/H+ exchanger is a ubiquitous, integral membrane protein involved in pH regulation. It removes intracellular acid, exchanging a proton for an extracellular sodium ion. There are seven known isoforms of this protein that are the products of distinct genes. The first isoform discovered (NHE1) is ubiquitously distributed throughout the plasma membrane of virtually all tissues. It plays many different physiological roles in mammals, including important functions in regulation of intracellular pH, in heart disease, and in cytoskeletal organization. The first 500 amino acids of the protein are believed to consist of 12 transmembrane helices, a membrane-associated segment, and two reentrant loops. A C-terminal regulatory domain of approximately 315 amino acids regulates the protein and mediates cytoskeletal interactions. Studies are underway to determine the amino acid residues important in NHE1 function. At present, it is clear that transmembrane segment IV is important in NHE1 function and that transmembrane segments VII and IX are also involved in transport. Further experiments are required to elucidate the mechanism of transport and regulation of this multifunctional protein.     

Functional importance of charged residues within the putative intracellular loops in pH regulation by Na+/ H+ exchanger NHE1

The plasma membrane Na+/H+ exchanger 1 is activated in response to various extrinsic factors, and this process is regulated by an intracellular pH-sensing mechanism. To identify the candidate residues responsible for intracellular pH regulation, we analyzed the functional properties of engineered Na+/H+ exchanger 1 mutants with charge-reversal mutations of charged residues located in the intracellular loops. Na+/H+ exchanger 1 mutants with mutations at 11 positions were well expressed in the plasma membrane, but that with E247R was not, suggesting that Glu247 is important for the functional expression of Na+/H+ exchanger 1. Charge-reversal mutations of Glu131 (E131R, E131K) and Arg327 (R327E) resulted in a shift in the intracellular pH dependence of the exchange activity measured by 22Na+ uptake to the acidic side, and it abolished the response to growth factors and a hyperosmotic medium; however, mutations of Asp448 (D448R) and Arg500 (R500E) slightly shifted it to the alkaline side. In E131R, in addition to the change in intracellular pH dependence, the affinities for extracellular Na+, Li+ and the inhibitor 5-(N-ethyl-N-isopropyl)amiloride significantly increased. Furthermore, charge-conserved mutation of E131 (E131D) was found to have no effect, whereas charge neutralization (E131Q) resulted in a slight acidic shift of exchange. These results support the view that the multiple charged residues identified in this study, along with several basic residues reported previously, participate in the regulation of the intracellular pH sensing of Na+/H+ exchanger 1. In addition, Glu131 may also be important for cation transport.     

The intracellular distal tail of the Na+/H+ exchanger NHE1 is intrinsically disordered: implications for NHE1 trafficking

Intrinsic disorder is important for protein regulation, yet its role in regulation of ion transport proteins is essentially uninvestigated. The ubiquitous plasma membrane carrier protein Na(+)/H(+) Exchanger isoform 1 (NHE1) plays pivotal roles in cellular pH and volume homeostasis, and its dysfunction is implicated in several clinically important diseases. This study shows, for the first time for any carrier protein, that the distal part of the C-terminal intracellular tail (the cdt, residues V686-Q815) from human (h) NHE1 is intrinsically disordered. Further, we experimentally demonstrated the presence of a similar region of intrinsic disorder (ID) in NHE1 from the teleost fish Pleuronectes americanus (paNHE1), and bioinformatic analysis suggested ID to be conserved in the NHE1 family. The sequential variation in structure propensity as determined by NMR, but not the amplitude, was largely conserved between the h- and paNHE1cdt. This suggests that both proteins contain molecular recognition features (MoRFs), i.e., local, transiently formed structures within an ID region. The functional relevance of the most conserved MoRF was investigated by introducing a point mutation that significantly disrupted the putative binding feature. When this mutant NHE1 was expressed in full length NHE1 in AP1 cells, it exhibited impaired trafficking to the plasma membrane. This study demonstrated that the distal regulatory domain of NHE1 is intrinsically disordered yet contains conserved regions of transient structure. We suggest that normal NHE1 function depends on a protein recognition element within the ID region that may be linked to NHE1 trafficking via an acidic ER export motif.     

Intravesicular pH changes in submitochondrial particles induced by monovalent cations: relationship to the Na+/H+ and K+/H+ antiporters

The fluorescence of internalized fluorescein isothiocyanate dextran has been used to monitor the intravesicular pH of submitochondrial particles (SMP). Respiring SMP maintain a steady-state delta pH (interior acid) that results from the inwardly directed H+ flux of respiration and an opposing passive H+ leak. Addition of K+, Na+, or Li+ to SMP results in a shift to a more alkaline interior pH (pHi) in both respiring and nonrespiring SMP. The K+-dependent change in pHi, like the K+/H+ antiport in intact mitochondria, is inhibited by quinine and by dicyclohexylcarbodiimide. The Na+-dependent reaction is only partially inhibited by these reagents. Both the Na+- and the K+-dependent pH changes are sensitive to amiloride derivatives. The Km for both Na+ and K+ is near 20 mM whereas that for Li+ is closer to 10 mM. The K+/H+ exchange reaction is only slightly inhibited by added Mg2+, but abolished when A23187 is added with Mg2+. The passive exchange is optimal at pHi 6.5 with either Na+ or K+, and cannot be detected above pHi of 7.2. Both the Na+/H+ and the K+/H+ exchange reactions are optimal at an external pH of 7.8 in respiring SMP (pHi 7.1). Valinomycin stimulates the K+-dependent pH change in nonrespiring SMP, as does nigericin. It is concluded that SMP show K+/H+ antiport activity with properties distinct from those of Na+/H+ antiport. However, the properties of the K+/H+ exchange do not correspond in all respects to those of the antiport in intact mitochondria. Donnan equilibria and parallel uniport pathways for H+ and cations appear to contribute to cation-dependent pH changes in SMP.     

Model structure of the Na+/H+ exchanger 1 (NHE1): functional and clinical implications

Eukaryotic Na(+)/H(+) exchangers are transmembrane proteins that are vital for cellular homeostasis and play key roles in pathological conditions such as cancer and heart diseases. Using the crystal structure of the Na(+)/H(+) antiporter from Escherichia coli (EcNhaA) as a template, we predicted the three-dimensional structure of human Na(+)/H(+) exchanger 1 (NHE1). Modeling was particularly challenging because of the extremely low sequence identity between these proteins, but the model structure is supported by evolutionary conservation analysis and empirical data. It also revealed the location of the binding site of NHE inhibitors; which we validated by conducting mutagenesis studies with EcNhaA and its specific inhibitor 2-aminoperimidine. The model structure features a cluster of titratable residues that are evolutionarily conserved and are located in a conserved region in the center of the membrane; we suggest that they are involved in the cation binding and translocation. We also suggest a hypothetical alternating-access mechanism that involves conformational changes.     

Comparative biology of the ubiquitous Na+/H+ exchanger, NHE1: lessons from erythrocytes

By virtue of their electroneutral exchange of intracellular H+ for extracellular Na+, the Na+/H+ exchangers (NHE1-NHE8) play a pivotal role in many physiological processes. This review focuses on the ubiquitous plasma membrane isoform, NHE1. Particular attention is given to the roles and regulation of NHE1 in erythrocytes, in their own right and as model systems, but pertinent findings from non-erythroid cells are also discussed. NHE1 plays a key role in the regulation of cell volume and pH, and consequently in the control of such diverse processes as blood O2/CO2 transport, and cell proliferation, motility, and survival. Disturbances in NHE1 function are involved in important pathological states such as hypoxic cell damage and cancer development. NHE1 has a predicted topology of 12 transmembrane domains, and a hydrophilic C-terminus thought to be the major site for NHE1 regulation. NHE1 is highly conserved throughout the vertebrate phylum, particularly in the transmembrane region and the proximal part of the C-terminus. In non-erythroid, and probably also in erythroid cells, this part of the hydrophilic C-terminus interacts with multiple binding partners important for NHE1 function. Erythrocyte NHE1s from mammalian, amphibian, and teleost species are activated by cell shrinkage, decreased pH(i), inhibition of Ser/Thr protein phosphatases, and activation of Ser/Thr protein kinases, i.e., many of the stimuli activating NHE1 in non-erythroid cells. In erythrocytes of many lower vertebrates, NHE1 is activated during hypoxia and is an important modulator of hemoglobin oxygen affinity. Sensitivity of NHE1 to oxygenation status has recently been described also in non-erythroid mammalian cells.     

Characterization of Na+/H+ Exchanger Isoform (NHE1, NHE2 and NHE3) Expression in Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. Na⁺/H⁺ exchange (NHE) is one of the major Na⁺ absorptive pathways in gallbladder. In this study, we measured gallbladder Na⁺/H⁺ exchange and characterized the NHE isoforms expressed in prairie dogs. Na⁺/H⁺ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na⁺ flux and apical ²²Na⁺ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal J ^Na _ms was higher than J ^Na _sm with J ^Na _net absorption. Mucosal DMA inhibited transepithelial Na⁺ flux in a dose-dependent fashion, causing J ^Na _ms equal to J ^Na _sm and blocking J ^Na _net absorption at 100 μm. Basal ²²Na⁺ uptake rate was 10.9 ± 1.0 μmol · cm⁻²· hr⁻¹ which was inhibited by ∼43% by mucosal DMA and ∼30% by mucosal HOE-694 at 100 μm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na⁺/H⁺ exchange accounts for a substantial fraction of gallbladder apical Na⁺ entry and most of net Na⁺ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na⁺ uptake and transepithelial Na⁺ absorption. Received: 9 February 2001/Revised: 11 April 2001     

Effect of thymoquinone on cytosolic pH and Na+/H+ exchanger activity in mouse dendritic cells

The anti-inflammatory Nigella sativa component thymoquinone compromises the function of dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. DC function is regulated by the Na(+)/H(+) exchanger (NHE), which is stimulated by lipopolysaccharides (LPS) and required for LPS-induced cell swelling, reactive oxygen species (ROS) production, TNF-α release and migration. Here we explored, whether thymoquinone influences NHE activity in DCs. To this end, bone marrow derived mouse DCs were treated with LPS in the absence and presence of thymoquinone (10 μM). Cytosolic pH (pH(i)) was determined from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNF-α production utilizing ELISA and DC migration with transwell migration assays. As a result, exposure of DCs to LPS (1 μg/ml) led within 4 hours to transient increase of NHE activity. Thymoquinone did not significantly modify cytosolic pH or cellular NHE activity in the absence of LPS, but abrogated the effect of LPS on NHE activity. Accordingly, in the presence of thymoquinone LPS-treatment resulted in cytosolic acidification. LPS further increased forward scatter and ROS formation, effects similarly abrogated by thymoquinone. Again, in the absence of LPS, thymoquinone did not significantly modify ROS formation and cell volume. LPS further triggered TNF-α release and migration, effects again blunted in the presence of thymoquinone. NHE1 inhibitor cariporide (10 μM) blunted LPS induced TNF-α release and migration. The effects of thymoquinone on NHE activity and migration were reversed upon treatment of the cells with t-butyl hydroperoxide (TBOOH, 5 μM). In conclusion, thymoquinone blunts LPS induced NHE activity, cell swelling, oxidative burst, cytokine release and migration of bone marrow derived murine dendritic cells. NHE inhibition may thus contribute to the antiinflammatory action of thymoquinone.     

Impaired gastric acid secretion in mice with a targeted disruption of the NHE4 Na+/H+ exchanger

The NHE4 Na+/H+ exchanger is abundantly expressed on the basolateral membrane of gastric parietal cells. To test the hypothesis that it is required for normal acid secretion, NHE4-null mutant (NHE4-/-) mice were prepared by targeted disruption of the NHE4 (Slc9a4) gene. NHE4-/- mice survived and appeared outwardly normal. Analysis of stomach contents revealed that NHE4-/- mice were hypochlorhydric. The reduction in acid secretion was similar in 18-day-old, 9-week-old, and 6-month-old mice, indicating that the hypochlorhydria phenotype did not progress over time, as was observed in mice lacking the NHE2 Na+/H+ exchanger. Histological abnormalities were observed in the gastric mucosa of 9-week-old NHE4-/- mice, including sharply reduced numbers of parietal cells, a loss of mature chief cells, increased numbers of mucous and undifferentiated cells, and an increase in the number of necrotic and apoptotic cells. NHE4-/- parietal cells exhibited limited development of canalicular membranes and a virtual absence of tubulovesicles, and some of the microvilli had centrally bundled actin. We conclude that NHE4, which may normally be coupled with the AE2 Cl-/HCO3- exchanger, is important for normal levels of gastric acid secretion, gastric epithelial cell differentiation, and development of secretory canalicular and tubulovesicular membranes.     

ATP dependence is not an intrinsic property of Na+/H+ exchanger NHE1: requirement for an ancillary factor

Na⁺/H⁺exchange is a passive process not requiring expenditure of metabolicenergy. Nevertheless, depletion of cellular ATP produces a markedinhibition of the antiport. No evidence has been found for directbinding of nucleotide to exchangers or alteration in their state ofphosphorylation, suggesting ancillary factors may be involved. Thispossibility was tested by comparing the activity of dog red blood cells(RBC) and their resealed ghosts. Immunoblotting experiments usingisoform-specific polyclonal and monoclonal antibodies indicated RBCmembranes expressNa⁺/H⁺exchanger isoform 1 (NHE1). In intact RBC, uptake ofNa⁺ was greatly stimulated whenthe cytosol was acidified. The stimulated uptake was largely eliminatedby amiloride and by submicromolar concentrations of the benzoylguanidinium compound HOE-694, consistent with mediation by NHE1.Although exchange activity could also be elicited by acidification inresealed ghosts containing ATP, the absolute rate of transport wasmarkedly diminished at comparable pH. Dissipation of the pH gradientwas ruled out as the cause of diminished transport rate in ghosts. Thiswas accomplished by a "pH clamping" procedure based on continuedexport of base equivalents by the endogenous anion exchanger. Theseobservations suggest a critical factor required to maintain optimalNa⁺/H⁺exchange activity is lost or inactivated during preparation of ghosts.Depletion of ATP, achieved by incubation with2-deoxy-D-glucose, inhibitedNa⁺/H⁺exchange in intact RBC, as reported for nucleated cells. In contrast, the rate of exchange was similar in control and ATP-depleted resealed ghosts. Interestingly, the residual rate ofNa⁺/H⁺exchange in ATP-depleted but otherwise intact cells was similar to thetransport rate of ghosts. Therefore, we tentatively conclude that fullactivation of NHE1 requires both ATP and an additional regulatoryfactor, which may mediate the action of the nucleotide. Ancillaryphosphoproteins or phospholipids or the kinases that mediate theirphosphorylation are likely candidates for the regulatory factor(s) thatis inactivated or missing in ghosts.

     

Topological analysis of NHE1, the ubiquitous Na+/H+ exchanger using chymotryptic cleavage

Proteases,glycosidases, and impermeant biotin derivatives were used incombination with antibodies to analyze the subcellular distribution andtransmembrane disposition of theNa⁺/H⁺exchanger NHE1. Both native human NHE1 in platelets and epitope-tagged rat NHE1 transfected into antiport-deficient cells were used for thesestudies. The results indicated that1) the entire population ofexchangers is present on the surface membrane of unstimulated platelets, ruling out regulation by recruitment of internal stores ofNHE1; 2) the putative extracellularloops near the NH₂ terminus areexposed to the medium and contain all the N- andO-linked carbohydrates;3) by contrast, the putativeextracellular loops between transmembrane domains 9-10 and11-12 are not readily accessible from the outside and may befolded within the protein, perhaps contributing to an aqueous iontransport pathway; 4) the extreme COOH terminus of the protein was found to be inaccessible toextracellular proteases, antibodies, and other impermeant reagents,consistent with a cytosolic localization; and5) detachment of ~150 amino acidsfrom the NH₂-terminal end of theprotein had little effect on the transport activity of NHE1.

     

Cannabinoids inhibit insulin secretion and cytosolic Ca2+ oscillation in islet beta-cells via CB1 receptors

Obesity is the main risk factor for the development of metabolic syndrome. Endogenous cannabinoids act on the cannabinoid type 1 (CB1) receptor, a GPCR, and stimulate appetite via central and peripheral actions, while blockade of CB1 receptor reduces body weight in humans. In this study, we aimed to explore a role of the peripheral endocannabinoid system in insulin secretion, which could be important in the metabolic effects of the cannabinoid-CB1 system. We found that mRNA for CB1 receptor, but not CB2 receptor, was expressed in mouse pancreatic islets using RT-PCR. Immunohistochemical study revealed that CB1 receptor was expressed in beta-cells. Furthermore, anandamide and a CB1 agonist, arachidonylcyclopropylamide (ACPA), inhibited glucose-induced insulin secretion from mouse pancreatic islets. Both anandamide and ACPA inhibited glucose-induced cytosolic Ca(2+) oscillation in mouse pancreatic beta-cells. These results demonstrate a novel peripheral action of cannabinoids to inhibit insulin secretion via CB1 receptors.