TNK cells (NKG2D<Superscript>+</Superscript> CD8<Superscript>+</Superscript> or CD4<Superscript>+</Superscript> T lymphocytes) in the control of human tumors

Innate and adaptive immune responses have many interactions that are regulated by the balance of signals initiated by a variety of activatory and inhibitory receptors. Among these, the NKG2D molecule was identified as expressed by T lymphocytes, including most CD8⁺ cells and a minority of CD4⁺ cells, designated TNK cells in this paper. Tumor cells may overexpress the stress-inducible NKG2D ligands (NKG2DLs: MICA/B, ULBPs) and the NKG2D signaling has been shown to be involved in lymphocyte-mediated anti-tumor activity. Aberrant expression of NKG2DLs by cancer cells, such as the release of soluble form of NKG2DLs, can lead to the impairment of these immune responses. Here, we discuss the significance of NKG2D in TNK-mediated anti-tumor activity. Our studies demonstrate that NKG2D⁺ T cells (TNK) are commonly recruited at the tumor site in melanoma patients where they may exert anti-tumor activity by engaging both TCR and NKG2D. Moreover, NKG2D and TCR triggering was also observed by peripheral blood derived T lymphocyte- or T cell clone-mediated tumor recognition, both in melanoma and colorectal cancer (CRC) patients. Notably, heterogeneous expression of NKG2DLs was found in melanoma and CRC cells, with a decrease of these molecules along with tumor progression. Therefore, through the mechanisms that govern NKG2D engagement in anti-tumor activity and the expression of NKG2DLs by tumor cells that still need to be dissected, we showed that NKG2D expressing TNK cells are a relevant T cell subtype for immunosurveillance of tumors and we propose that new immunotherapeutic interventions for cancer patients should be aimed also at enhancing NKG2DLs expression by tumor cells. This paper is a focused research review based on a presentation given at the sixth annual meeting of the Association for Immunotherapy of Cancer (CIMT), held in Mainz, Germany, 15–16 May 2008.     

The control of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>Foxp3<Superscript>+</Superscript>regulatory T cell survival

   

CD4⁺CD25⁺Foxp3⁺ regulatory T (T_reg) cells are believed to play an important role in suppressing autoimmunity and maintaining peripheral tolerance. How their survival is regulated in the periphery is less clear. Here we show that T_reg cells express receptors for gamma chain cytokines and are dependent on an exogenous supply of these cytokines to overcome cytokine withdrawal apoptosis in vitro. This result was validated in vivo by the accumulation of T_reg cells in Bim^-/- and Bcl-2 tg mice which have arrested cytokine deprivation apoptosis. We also found that CD25 and Foxp3 expression were down-regulated in the absence of these cytokines. CD25⁺ cells from Scurfy mice do not depend on cytokines for survival demonstrating that Foxp3 increases their dependence on cytokines by suppressing cytokine production in T_reg cells. Our study reveals that the survival of T_reg cells is strictly dependent on cytokines and cytokine producing cells because they do not produce cytokines. Our study thus, demonstrates that different gamma chain cytokines regulate T_reg homeostasis in the periphery by differentially regulating survival and proliferation. These findings may shed light on ways to manipulate T_reg cells that could be utilized for their therapeutic applications.     

Effects of ouabain on proliferation of human endothelial cells correlate with Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity and intracellular ratio of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript>

Side-by-side with inhibition of the Na⁺,K⁺-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na⁺ and K⁺ ratio ([Na⁺]_i/[K⁺]_i). Thus, we compared the doseand time-dependences of the effect of ouabain on intracellular [Na⁺]_i/[K⁺]_i ratio, Na⁺,K⁺-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na⁺]_i/[K⁺]_i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na⁺,K⁺-ATPase activity by 25-30%, as measured by the rate of ⁸⁶Rb⁺ influx. Higher ouabain concentrations inhibited Na⁺,K⁺-ATPase, increased [Na⁺]_i/[K⁺]_i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na⁺]_i/[K⁺]_i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na⁺]_i/[K⁺]_i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na⁺ and K⁺ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na⁺,K⁺-ATPase and decrease in the [Na⁺]_i/[K⁺]_i ratio.     

CD133<Superscript>+</Superscript>CD44<Superscript>+</Superscript> subgroups may be human small intestinal stem cells

The identification and separation of small intestinal epithelial stem cells are still on the preliminary stage. In this study, we planned to utilize immunohistochemistry, fluorescence-activated cell sorting (FACS) and RT-PCR to investigate the possibility of CD133 and CD44 as markers of human small intestinal epithelial stem cells. The expressions of CD133, CD44 and Lgr5 were studied by immunohistochemistry. Four subgroups of CD133⁺CD44⁺, CD133⁺CD44⁻, CD133⁻CD44⁺, CD133⁻CD44⁻ were sorted out through FACS and the expression level of Lgr5 gene was measured by RT-PCR and polyacrylamide gel electropheresis (PAGE) with sliver stained. Ten cases of samples were available for analyzing. By immunohistochemical staining, few cells with positive expressions of CD133, CD44 and Lgr5 were distributed in the bottom of crypts with the expression locations somewhat overlapped. The average percentage of CD133⁺CD44⁺ cells was 0.0580 ± 0.0403%, while the corresponding contents of CD133⁺CD44⁻ cells, CD133⁻CD44⁺ cells and CD133⁻CD44⁻ cells were 0.4000 ± 0.1225%, 0.7000 ± 0.2646% and 76.5600 ± 3.5529% respectively. Ten times of positive expressions of Lgr5 were detected in the CD133⁺CD44⁺ groups, while 9/10, 8/10 and 4/10 times for CD133⁺CD44⁻, CD133⁻CD44⁺ and CD133⁻CD44⁻ subgroups respectively. With the help of Quantityone 4.62 software, the densities of corresponding place to Lgr5 and reference gene were obtained. The density ratios of corresponding place to Lgr5 to reference gene were significant difference between subgroups (P < 0.001). By means of LSD method, the density ratios in CD133⁺CD44⁺ subgroups had statistical differences from the other subgroups (P < 0.05). We concluded CD133⁺CD44⁺ cells may be human small intestinal epithelial stem cells, which need further researches to confirm.     

A possible mechanism for low affinity of silkworm Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase for K<Superscript>+</Superscript>

The affinity for K⁺ of silkworm nerve Na⁺/K⁺-ATPase is markedly lower than that of mammalian Na⁺/K⁺-ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K⁺ affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na⁺/K⁺-ATPase α and β subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and β subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na⁺/K⁺-ATPase α1 subunit. On the other hand, the amino acid identity of the β subunit with mammalian counterparts was as low as 30%. Cloned α and β cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na⁺/K⁺-ATPase. Na⁺/K⁺-ATPase expressed in the cultured cells showed a low affinity for K⁺ and a high affinity for Na⁺, characteristic of the silkworm nerve Na⁺/K⁺-ATPase. These results suggest that the β subunit is responsible for the affinity for K⁺ of Na⁺/K⁺-ATPase.     

Sepsis correlated with increased erythrocyte Na<Superscript>+</Superscript> content and Na<Superscript>+</Superscript>-K<Superscript>+</Superscript> pump activity

The aims of the present study were twofold: (1) simultaneous determinations of Na(+) transport parameters of erythrocytes from 40 healthy donors and 28 septic patients as assessed by a score of severity of sepsis (SSS), and (2) examination of the correlation between the SSS and specific Na(+) transport abnormalities. Erythrocytes were obtained and loaded with different ionic compositions and cellular Na(+) contents before determination of the near-maximal Na(+) pump rate (Vmax), the physiological extrusion rate of Na(+) (v) and the number of ouabain-binding sites (Bmax). In erythrocytes from septic patients, the cellular Na(+) content was 28% higher (p < 0.001), with no differences in water content compared to erythrocytes from healthy donors. This elevated Na(+) content was accompanied by significantly higher values for Vmax (43%), v (24%) and Bmax (48%) of the Na(+) pump in septic erythrocytes. Moreover, significant positive correlations existed between Vmax and SSS (p = 0.028) and between cellular Na(+) content and SSS (p = 0.005). These data suggest that during sepsis, membrane alterations occur and result in an increased cellular Na(+) content. Active Na(+) transport (Vmax and v) was significantly stimulated, possibly as a consequence of a secondary response to the elevated Na(+) of cells. Both cellular Na(+) and Vmax correlated well with the severity of sepsis, suggesting that these altered transport parameters may reflect the progress of sepsis.     

Primed tumor-reactive multifunctional CD62L<Superscript>+</Superscript> human CD8<Superscript>+</Superscript> T cells for immunotherapy

T cell-mediated immunotherapy against malignancies has been shown to be effective for certain types of cancer. However, ex vivo expansion of tumor-reactive T cells has been hindered by the low precursor frequency of such cells, often requiring multiple rounds of stimulation, resulting in full differentiation, loss of homing receptors and potential exhaustion of the expanded T cells. Here, we show that when using highly purified naïve CD8⁺ T cells, a single stimulation with peptide-pulsed, IFNγ/LPS-matured dendritic cells in combination with the sequential use of IL-21, IL-7 and IL-15 is sufficient for extensive expansion of antigen-specific T cells. Short-term expanded T cells were tumor-reactive, multifunctional and retained a central-memory-like phenotype (CD62L⁺, CCR7⁺, CD28⁺). The procedure is highly reproducible and robust as demonstrated for different healthy donors and for cancer patients. Such short-term tumor-antigen-primed, multifunctional T cells may therefore serve as a platform to target different malignancies accessible to immunotherapy.     

Salt tolerance of barley: Relations between expression of isoforms of vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript>-antiporter and <Superscript>22</Superscript>Na<Superscript>+</Superscript> accumulation

Two barley cultivars (Hordeum vulgare L., cvs. Elo and Belogorskii) differing in salt tolerance were used to study ²²Na⁺ uptake, expression of three isoforms of the Na⁺/H⁺ antiporter HvNHX1-3, and the cellular localization of these isoforms in the elongation zone of seedling roots. During short (1 h) incubation, seedling roots of both cultivars accumulated approximately equal quantities of ²²Na⁺. However, after 24-h incubation the content of ²²Na⁺ in roots of a salt-tolerant variety Elo was 40% lower than in roots of the susceptible variety Belogorskii. The content of ²²Na⁺ accumulated in shoots of cv. Elo after 24-h incubation was 6.5 times lower than in shoots of cv. Belogorskii and it was 4 times lower after the salt stress treatment. The cytochemical examination revealed that three proteins HvNHX1-3 are co-localized in the same cells of almost all root tissues; these proteins were present in the tonoplast and prevacuolar vesicles. Western blot analysis of HvNHX1-3 has shown that the content of isoforms in vacuolar membranes increased in response to salt stress in seedling roots and shoots of both cultivars, although the increase was more pronounced in the tolerant cultivar. The content of HvNHX1 in the seedlings increased in parallel with the enhanced expression of HvNHX1, whereas the increase in HvNHX2 and HvNHX3 protein content was accompanied by only slight changes in expression of respective genes. The results provide evidence that salt tolerance of barley depends on plant ability to restrict Na⁺ transport from the root to the shoot and relies on regulatory pathways of HvNHX1-3 expression in roots and shoots during salt stress.     

Changes in Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity and Alpha 3 Subunit Expression in CNS After Administration of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitors

The expression of Na⁺, K⁺-ATPase α3 subunit and synaptosomal membrane Na⁺, K⁺-ATPase activity were analyzed after administration of ouabain and endobain E, respectively commercial and endogenous Na⁺, K⁺-ATPase inhibitors. Wistar rats received intracerebroventricularly ouabain or endobain E dissolved in saline solution or Tris–HCl, respectively or the vehicles (controls). Two days later, animals were decapitated, cerebral cortex and hippocampus removed and crude and synaptosomal membrane fractions were isolated. Western blot analysis showed that Na⁺, K⁺-ATPase α3 subunit expression increased roughly 40% after administration of 10 or 100 nmoles ouabain in cerebral cortex but remained unaltered in hippocampus. After administration of 10 μl endobain E (1 μl = 28 mg tissue) Na⁺, K⁺-ATPase α3 subunit enhanced 130% in cerebral cortex and 103% in hippocampus. The activity of Na⁺, K⁺-ATPase in cortical synaptosomal membranes diminished or increased after administration of ouabain or endobain E, respectively. It is concluded that Na⁺, K⁺-ATPase inhibitors modify differentially the expression of Na⁺, K⁺-ATPase α3 subunit and enzyme activity, most likely involving compensatory mechanisms.     

LPS administration increases CD11b<Superscript>+</Superscript> c-Fms<Superscript>+</Superscript> CD14<Superscript>+</Superscript> cell population that possesses osteoclast differentiation potential in mice

Osteoclasts are multinucleated giant cells that originate from a monocyte/macrophage lineage, and are involved in the inflammatory bone destruction accompanied by periodontitis. Recent studies have shown that osteoclast precursors reside not only in the bone marrow, but also in the peripheral blood and spleen, though the precise characteristics of each precursor have not been analyzed. We hypothesized that the number of osteoclast precursors in those tissues may increase under pathological conditions and contribute to osteoclast formation in vivo in a mouse model. To test this hypothesis, we attempted to identify cell populations that possess osteoclast differentiation potential in the bone marrow, spleen, and blood by analyzing macrophage/monocyte-related cell surface markers such as CD11b, CD14, and colony-stimulating factor-1 receptor (c-Fms). In the bone marrow, the CD11b^? cell population, but not the CD11b⁺ cell population, differentiated into osteoclasts in the presence of receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor. On the other hand, in the spleen and blood, CD11b⁺ cells differentiated into osteoclasts. Interestingly, lipopolysaccharide (LPS) administration to the mice dramatically increased the proportion of CD11b⁺ c-Fms⁺ CD14⁺ cells, which differentiated into osteoclasts, in the bone marrow and spleen. These results suggest that LPS administration increases the proportion of a distinct cell population expressing CD11b⁺, c-Fms⁺, and CD14⁺ in the bone marrow and spleen. Thus, these cell populations are considered to contribute to the increase in osteoclast number during inflammatory bone destruction such as periodontitis.     

Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> and K<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporters AtNHX1 and AtNHX3 from <Emphasis Type="Italic">Arabidopsis</Emphasis> improve salt and drought tolerance in transgenic poplar

The tonoplast and plasma membrane localized sodium (potassium)/proton antiporters have been shown to play an important role in plant resistance to salt stress. In this study, AtNHX1 and AtNHX3, two tonoplast Na⁺(K⁺)/H⁺ antiporter encoding genes from Arabidopsis thaliana, were expressed in poplar to investigate their biological functions in the resistance to abiotic stresses in woody plants. Transgenic poplar plants expressing either gene exhibited increased resistance to both salt and water-deficit stresses. Compared to the wild type (WT) plants, transgenic plants accumulated more sodium and potassium ions in the presence of 100 mM NaCl and showed reduced electrolyte leakage in the leaves under water stress. Furthermore, the proton-translocating and cation-dependent H⁺ (Na⁺/H⁺ or K⁺/H⁺) exchange activities in the tonoplast vesicles isolated from the leaves of transgenic plants were higher than in those isolated from WT plants. Therefore, constitutive expression of either AtNHX1 or AtNHX3 genetically modified the salt and water stress tolerance of transgenic poplar plants, providing a potential tool for engineering tree species with enhanced resistance to multiple abitotic stresses.     

Molecular and functional analyses of rice NHX-type Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter genes

We previously cloned a vacuolar Na⁺/H⁺ antiporter gene (OsNHX1) from rice (Oryza sativa). Here we identified four additional NHX-type antiporter genes in rice (OsNHX2 through OsNHX5) and performed molecular and functional analyses of those genes. The exon–intron structure of the OsNHX genes and the phylogenetic tree of the OsNHX proteins suggest that the OsNHX proteins are categorized into two subgroups (OsNHX1 through OsNHX4 and OsNHX5). OsNHX1, OsNHX2, OsNHX3, and OsNHX5 can suppress the Na⁺, Li⁺, and hygromycin sensitivity of yeast nhx1 mutants and their sensitivity to a high K⁺ concentration. The expression of OsNHX1, OsNHX2, OsNHX3, and OsNHX5 is regulated differently in rice tissues and is increased by salt stress, hyperosmotic stress, and ABA. When we studied the expression of β-glucuronidase (GUS) driven by either the OsNHX1 or the OsNHX5 promoter, we observed activity in the stele, the emerging part of lateral roots, the vascular bundle, the water pore, and the basal part of seedling shoots with both promoters. In addition, each promoter had a unique expression pattern. OsNHX1 promoter–GUS activity only was localized to the guard cells and trichome, whereas OsNHX5 promoter–GUS activity only was localized to the root tip and pollen grains. Our results suggest that the members of this gene family play important roles in the compartmentalization into vacuoles of the Na⁺ and K⁺ that accumulate in the cytoplasm and that the differential regulation of antiporter gene expression in different rice tissues may be an important factor determining salt tolerance in rice.     

Identity of mysterious CD4<Superscript>+</Superscript>CD25<Superscript>-</Superscript>Foxp3<Superscript>+</Superscript>cells in systemic lupus erythematosus

Various abnormalities in CD4⁺CD25⁺ regulatory T cells (Tregs) in systemic lupus erythematosus (SLE) include increased Foxp3⁺ cells that are CD25 negative. Barring methodological technical factors, these cells could be atypical Tregs or activated non-Treg CD4⁺ cells that express Foxp3. Two groups have reached opposite conclusions that could possibly reflect the subjects studied. One group studied untreated new-onset SLE and suggested that these T cells were mostly CD25^-Foxp3⁺ non-Tregs. The other group studied patients with long-standing disease and suggested that these cells are mostly dysfunctional Tregs. A third group reported increased Foxp3⁺CD4⁺CD25^dim rather than CD25^- cells in active SLE and these were also non-Tregs. Thus, it is likely that not all Foxp3⁺ T cells in SLE have protective suppressive activity.     

Simultaneous ex vivo quantification of antigen-specific CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cell responses using in vitro transcribed RNA

Assessment of antigen-specific T-cell responses has been greatly facilitated by development of ELISPOT and intracellular cytokine flow cytometry (CFC) assays. The use of autologous antigen presenting cells transfected with in vitro transcribed RNA as stimulators allows in principle quantification of antigen-specific T-cells independent of the knowledge of the epitopes. We describe here a cytokine secretion assay that enables simultaneous assessment of both antigen-specific CD4+ as well as CD8+ T-cells directly from clinical samples without the need for generation of dendritic cells. To this aim, bulk PBMCs were electroporated with RNA encoding the antigen fused to trafficking signal sequences derived from a MHC class I molecule and used as stimulators. With human cytomegalovirus (HCMV) phosphoprotein 65 (pp65) as antigen we show that for measuring ex vivo T-cell responses in ELISPOT and CFC such stimulators are superior or at least equivalent to a pool of overlapping peptides representing the entire pp65 sequence as well as to untagged pp65 encoding RNA. This approach avoids the time consuming generation of dendritic cells as immune stimulators and, in particular when used in the context of the CFC, is robust, broadly applicable and fast.     

Regulation of durum wheat Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger TdSOS1 by phosphorylation

We have identified a plasma membrane Na⁺/H⁺ exchanger from durum wheat, designated TdSOS1. Heterologous expression of TdSOS1 in a yeast strain lacking endogenous Na⁺ efflux proteins showed complementation of the Na⁺- and Li⁺-sensitive phenotype by a mechanism involving cation efflux. Salt tolerance conferred by TdSOS1 was maximal when co-expressed with the Arabidopsis protein kinase complex SOS2/SOS3. In vitro phosphorylation of TdSOS1 with a hyperactive form of the Arabidopsis SOS2 kinase (T/DSOS2∆308) showed the importance of two essential serine residues at the C-terminal hydrophilic tail (S1126, S1128). Mutation of these two serine residues to alanine decreased the phosphorylation of TdSOS1 by T/DSOS2∆308 and prevented the activation of TdSOS1. In addition, deletion of the C-terminal domain of TdSOS1 encompassing serine residues at position 1126 and 1128 generated a hyperactive form that had maximal sodium exclusion activity independent from the regulatory SOS2/SOS3 complex. These results are consistent with the presence of an auto-inhibitory domain at the C-terminus of TdSOS1 that mediates the activation of TdSOS1 by the protein kinase SOS2. Expression of TdSOS1 mRNA in young seedlings of the durum wheat variety Om Rabia3, using different abiotic stresses (ionic and oxidative stress) at different times of exposure, was monitored by RT–PCR.     

Different domain organization of two main conformational states of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase

The Na⁺/K⁺-ATPase generates an electrochemical gradient of Na⁺ and K⁺, which is necessary for the functioning of animal cells. During the catalytic act, the enzyme passes through two principal conformational states, E₁ and E₂. To assess the domain organization of the protein in these conformations, thermal denaturation of Na⁺/K⁺-ATPases from duck salt gland and from rabbit kidney has been studied in the absence and in the presence of Na⁺ or K⁺, which induce the transition to E₁ or E₂. The melting curves for the ion-free forms of the two ATPases have different shapes: the rabbit protein shows one transition at 56.1°C, whereas the duck protein shows two transitions, at 49.8 and 56.9°C. Addition of Na⁺ or K⁺ ions abolishes the difference in thermal behavior between these enzymes, but through opposite effects. The melting curves for the E₂ conformation (K⁺ bound) in both cases exhibit a single peak of heat absorption at ∼63°C. For the E₁ conformation (Na⁺ bound), each melting curve has three peaks, indicating denaturation of three domains. The difference in the domain organization of Na⁺/K⁺-ATPase in the E1 and E2 states may account for the different sensitivity to temperature, proteolysis, and oxidative stress observed for the two enzyme conformations.     

Functional Characterization of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchangers in Primary Cultures of Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. We previously reported Na⁺/H⁺ exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I_sc, (11.6 ± 0.5 µA · cm^–2), potential differences, V_t (2.1 ± 0.2 mV), and resistance, R_t (169 ± 12 · cm²). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable ²²Na⁺ uptake under a H⁺ gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of ²²Na⁺ uptake was 21.4 ± 1.3 nmol · mg prot^–1 · min^–1, of which 9.5 ± 0.7 (~45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for ~6%, ~66% and ~28% of GBECs total NHE activity, respectively. GBECs exhibited saturable NHE kinetics (V_max 9.2 ± 0.3 nmol · mg prot^–1 · min^–1; K_m 11.4 ± 1.4 mM Na⁺). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na⁺ transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.     

Glutamatergic hypothesis of schizophrenia: involvement of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-dependent glutamate transport

Summary Hypothetical model based on deficient glutamatergic neurotransmission caused by hyperactive glutamate transport in astrocytes surrounding excitatory synapses in the prefrontal cortex is examined in relation to the aetiology of schizophrenia. The model is consistent with actions of neuroleptics, such as clozapine, in animal experiments and it is strongly supported by recent findings of increased expression of glutamate transporter GLT in prefrontal cortex of patients with schizophrenia. It is proposed that mechanisms regulating glutamate transport be investigated as potential targets for novel classes of neuroactive compounds with neuroleptic characteristics. Development of new efficient techniques designed specifically for the purpose of studying rapid activity-dependent translocation of glutamate transporters and associated molecules such as Na⁺, K⁺-ATPase is essential and should be encouraged.