Short-term effect on intestinal epithelial Na(+)/H(+) exchanger by Gi(alpha1,2)-coupled 5-HT(1A) and G(q/11)-coupled 5-HT(2) receptors

The present study evaluated the effect of 5-hydroxytryptamine (5-HT) on intestinal Na(+)/H(+) exchanger (NHE) activity and the cellular signaling pathways involved in T84 cells. T84 cells express endogenous NHE1 and NHE2 proteins, detected by immunoblotting, but not NHE3. The rank order for inhibition of NHE activity in acid-loaded T84 cells was 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; IC(50)=519 [465, 579] nM)>cariporide (IC(50)=630 [484, 819] nM)>amiloride (IC(50)=19 [16, 24] microM); the NHE3 inhibitor S3226 was found to be devoid of effect. This different inhibitory sensitivity indicates that both NHE1 and NHE2 isoforms may play an active role in Na(+)-dependent intracellular pH (pH(i)) recovery in T84 cells. Short-term exposure (0.5 h) of T84 cells to 5-HT increased NHE activity in a concentration-dependent manner. The stimulation induced by 5-HT (30 microM) was partially inhibited by both WAY 100135 (300 nM) and ketanserin (300 nM), antagonists of 5-HT(1A) and 5-HT(2) receptors, respectively. NHE activity was significantly increased by 8-OH-DPAT and alpha-methyl-5-HT, agonists of, respectively, 5-HT(1A) and 5-HT(2) receptors. An incubation of T84 cells with anti-G(s) and anti-G(beta) antibodies complexed with lipofectin did not prevent the 5-HT-induced stimulation of NHE activity. Overnight treatment with anti-G(ialpha1,2) and anti-G(q/11) antibodies complexed with lipofectin blocked the stimulatory effect induced by 8-OH-DPAT and alpha-methyl-5-HT, respectively. It is concluded that in T84 cells 5-HT enhances intestinal NHE activity through stimulation of G(ialpha1,2)-coupled 5-HT(1A) and G(q/11)-coupled 5-HT(2) receptors.     

Combined blockade of the Na+ channel and the Na+/H+ exchanger virtually prevents ischemic Na+ overload in rat hearts

Blocking either the Na⁺ channel or the Na⁺/H⁺ exchanger (NHE) has been shown to reduce Na⁺ and Ca²⁺ overload during myocardial ischemia and reperfusion, respectively, and to improve post-ischemic contractile recovery. The effect of combined blockade of both Na⁺ influx routes on ionic homeostasis is unknown and was tested in this study. [Na⁺]_i, pH_i and energy-related phosphates were measured using simultaneous ²³Na- and ³¹P-NMR spectroscopy in isolated rat hearts. Eniporide (3 μM) and/or lidocaine (200 μM) were administered during 5 min prior to 40 min of global ischemia and 40 min of drug free reperfusion to block the NHE and the Na⁺ channel, respectively. Lidocaine reduced the rise in [Na⁺]_i during the first 10 min of ischemia, followed by a rise with a rate similar to the one found in untreated hearts. Eniporide reduced the ischemic Na⁺ influx during the entire ischemic period. Administration of both drugs resulted in a summation of the effects found in the lidocaine and eniporide groups. Contractile recovery and infarct size were significantly improved in hearts treated with both drugs, although not significantly different from hearts treated with either one of them.     

Different mechanisms involved in apoptosis following exposure to benzo[a]pyrene in F258 and Hepa1c1c7 cells

The present study compares and elucidates possible mechanisms why B[a]P induces different cell signals and triggers apparently different apoptotic pathways in two rather similar cell lines (hepatic epithelial cells of rodents). The rate and maximal capacity of metabolic activation, as measured by the formation of B[a]P-tetrols and B[a]P-DNA adducts, was much higher in mouse hepatoma Hepa1c1c7 cells than in rat liver epithelial F258 cells due to a higher induced level of cyp1a1. B[a]P increased intracellular pH in both cell lines, but this change modulated the apoptotic process only in F258 cells. In Hepa1c1c7 cells reactive oxygen species (ROS) production appeared to be a consequence of toxicity, unlike F258 cells in which it was an initial event. The increased mitochondrial membrane potential found in F258 cells was not observed in Hepa1c1c7 cells. Surprisingly, F258 cells cultured at low cell density were somewhat more sensitive to low (50nM) B[a]P concentrations than Hepa1c1c7 cells. This could be explained partly by metabolic differences at low B[a]P concentrations. In contrast to the Hepa1c1c7 model, no activation of cell survival signals including p-Akt, p-ERK1/2 and no clear inactivation of pro-apoptotic Bad was observed in the F258 model following exposure to B[a]P. Another important difference between the two cell lines was related to the role of Bax and cytochrome c. In Hepa1c1c7 cells, B[a]P exposure resulted in a "classical" translocation of Bax to the mitochondria and release of cytochrome c, whereas in F258 cells no intracellular translocation of these two proteins was seen. These results suggest that the rate of metabolism of B[a]P and type of reactive metabolites formed influence the resulting balance of pro-apoptotic and anti-apoptotic cell signaling, and hence the mechanisms involved in cell death and the chances of more permanent genetic damage.     

IRBIT: A regulator of ion channels and ion transporters

IRBIT (also called AHCYL1) was originally identified as a binding protein of the intracellular Ca^2 + channel inositol 1,4,5-trisphosphate (IP₃) receptor and functions as an inhibitory regulator of this receptor. Unexpectedly, many functions have subsequently been identified for IRBIT including the activation of multiple ion channels and ion transporters, such as the Na⁺/HCO₃⁻ co-transporter NBCe1-B, the Na⁺/H⁺ exchanger NHE3, the Cl⁻ channel cystic fibrosis transmembrane conductance regulator (CFTR), and the Cl⁻/HCO₃⁻ exchanger Slc26a6. The characteristic serine-rich region in IRBIT plays a critical role in the functions of this protein. In this review, we describe the evolution, domain structure, expression pattern, and physiological roles of IRBIT and discuss the potential molecular mechanisms underlying the coordinated regulation of these diverse ion channels/transporters through IRBIT. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Sphingolipid regulation of ezrin,radixin, and moesin proteins family: Implications for cell dynamics

A key but poorly studied domain of sphingolipid functions encompasses endocytosis, exocytosis, cellular trafficking, and cell movement. Recently, the ezrin, radixin and moesin (ERM) family of proteins emerged as novel potent targets regulated by sphingolipids. ERMs are structural proteins linking the actin cytoskeleton to the plasma membrane, also forming a scaffold for signaling pathways that are used for cell proliferation, migration and invasion, and cell division. Opposing functions of the bioactive sphingolipid ceramide and sphingosine-1-phosphate (S1P), contribute to ERM regulation. S1P robustly activates whereas ceramide potently deactivates ERM via phosphorylation/dephosphorylation, respectively. This recent dimension of cytoskeletal regulation by sphingolipids opens up new avenues to target cell dynamics, and provides further understanding of some of the unexplained biological effects mediated by sphingolipids. In addition, these studies are providing novel inroads into defining basic mechanisms of regulation and action of bioactive sphingolipids. This review describes the current understanding of sphingolipid regulation of the cytoskeleton, it also describes the biologies in which ERM proteins have been involved, and finally how these two large fields have started to converge. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Direct catalytic electrochemistry of sulfite dehydrogenase: Mechanistic insights and contrasts with related Mo enzymes

Under hydrodynamic electrochemical conditions with slow cyclic voltammetry sweep rates we have been able to probe catalytic events at the molybdenum active site of sulfite dehydrogenase (SDH) from Starkeya novella adsorbed on an edge plane graphite electrode within a polylysine film. The electrochemically driven catalytic behaviour of SDH mirrors that seen in solution assays suggesting that the adsorbed enzyme retains its native activity. However, at high sulfite concentrations, the voltammetric waveform transforms from the expected sigmoidal profile to a peak-shaped response, similar to that reported for the molybdenum enzymes DMSO reductase and nitrate reductase (NarGHI and NapAB) where a redox reaction at the active site has been associated with a switch to lower activity at high overpotentials. This is the first time a similar phenomenon has been observed in a Mo-containing oxidase/dehydrogenase, which raises a number of interesting mechanistic problems. The potential at which the activity of SDH becomes attenuated only emerges at saturating substrate conditions and occurs at a potential (ca. + 320mV vs NHE) well removed from any known redox couple in the enzyme. These results cannot be explained by the same mechanism adopted for DMSO reductase and nitrate reductase catalysis.     

Protons extruded by NHE1: digestive or glue?

Many physiological and pathophysiological processes, such as embryogenesis, immune defense, wound healing, or metastasis, are based on cell migration and invasion. The activity of the ubiquitously expressed NHE1 isoform of the plasma membrane Na(+)/H(+) exchanger is one of the requirements for directed locomotion of migrating cells. The mechanisms by which NHE1 is involved in cell migration are multiple. NHE1 contributes to cell migration by affecting the cell volume, by regulating the intracellular pH and thereby the assembly and activity of cytoskeletal elements, by anchoring the cytoskeleton to the plasma membrane, by the organization of signal transduction and by regulating gene expression. The present review focuses on two additional, extracellular mechanisms by which NHE1 activity contributes to cell migration and invasion. Protons extruded by the NHE1 lead to local, extracellular acidification which, on the one hand, can create pH optima needed for the activity of proteinases at invadopodia/podosomes necessary for extracellular matrix digestion and, on the other hand, facilitates cell/matrix interaction and adhesion at the cell front.     

Abnormal Rab11‐Rab8‐vesicles cluster in enterocytes of patients with microvillus inclusion disease

Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by accumulation of vesiculo‐tubular endomembranes in the subapical cytoplasm of enterocytes, historically termed “secretory granules.” However, neither their identity nor pathophysiological significance is well defined. Using immunoelectron microscopy and tomography, we studied biopsies from MVID patients (3× Myosin 5b mutations and 1× Syntaxin3 mutation) and compared them to controls and genome‐edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from 2 patients with novel Myosin 5b mutations and typical clinical symptoms showed unusual ultrastructural phenotypes: aberrant subapical vesicles and tubules were prominent in the enterocytes, though other histological hallmarks of MVID were almost absent (ectopic intra‐/intercellular microvilli, brush border atrophy). We identified these enigmatic vesiculo‐tubular organelles as Rab11‐Rab8‐positive recycling compartments of altered size, shape and location harboring the apical SNARE Syntaxin3, apical transporters sodium‐hydrogen exchanger 3 (NHE3) and cystic fibrosis transmembrane conductance regulator. Our data strongly indicate that in MVID disrupted trafficking between cargo vesicles and the apical plasma membrane is the primary cause of a defect of epithelial polarity and subsequent facultative loss of brush border integrity, leading to malabsorption. Furthermore, they support the notion that mislocalization of transporters, such as NHE3 substantially contributes to the reported sodium loss diarrhea.      

A Toxoplasma gondii protein with homology to intracellular type Na/H exchangers is important for osmoregulation and invasion

The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na⁺/H⁺ exchanger in T. gondii, TgNHE3, and its co-localization with the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca²⁺ concentration [Ca²⁺]_i, and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.