Cl/HCO3 exchange in the basolateral membrane domain of rat jejunal enterocyte

Summary Basolateral membrane vesicles isolated from rat jejunal enterocyte and well purified from brush border contamination were tested to examine Cl and HCO₃ movements. Uptake experiments provided no evidence for a coupling between Na and HCO₃ fluxes; K–HCO₃ and K–Cl cotransports also could be excluded. Transport studies revealed the presence of a Cl/HCO₃ exchanger accepting other anions and inhibitable by the disulfonic stilbenes SITS and DIDS. We can exclude that the evidenced HCO₃-dependent Cl uptake is due to brush border contamination, since in jejunal brush border membranes this mechanism, if present, has a very low transport rate. Besides the Cl/HCO₃ antiporter, a Cl-conductive pathway seems to exist in jejunal basolateral membranes.     

Basolateral Cl/HCO3 exchange in rat jejunum: The effect of sodium

Basolateral membrane vesicles isolated from rat jejunum were used to characterize a Cl/HCO₃ exchange mechanism previously evidenced. Cl uptake experiments provided no evidence for Cl/OH countertransport, confirming anyhow the presence of Cl/HCO₃ antiport, which was inhibited by 2 mm furosemide and unaffected by 2 mm amiloride. An outwardly directed Na gradient stimulated Cl uptake and this effect was increased if Na was present at both vesicle surfaces. To investigate the mechanism of coupling between Na and the transport protein, we performed Na uptake experiments. Na uptake was unaffected by cis-bicarbonate and trans-Cl gradients; the reversal of anion gradients was still ineffective. Similar results were obtained when a pH difference across the membrane vesicles was imposed. This study seems to suggest that Na is not transported by the Cl/HCO₃ exchanger and that another mode of Na dependence must be taken into account.     

Interaction between Na ions and the Cl/HCO3 exchanger in basolateral membranes from rat jejunum

The jejunal basolateral Cl/HCO₃ exchanger is modulated by two Na-dependent regulatory sites located on the inner and outer membrane surfaces. The aim of this work was to focus on the interaction between the anion exchanger and intracellular or extracellular sodium. Uptake studies, performed using basolateral membrane vesicles, provided kinetic parameters as a function of outside or inside Na concentration. The intracellular Na-sensitive modifier site seems to be primarily involved in the modulation of the Cl/HCO₃ exchanger.     

Modulation of the frequency of glucose-dependent bursts of electrical activity by HCO3/CO2 in rodent pancreatic B-cells: experimental and theoretical results

The burst pattern of electrical activity recorded from pancreatic B-cells in response to 11 mM glucose shows a large islet to islet variability. The relationship between burst frequency and glucose sensing (the threshold for electrical activity and the graded increase in electrical response to glucose, i.e. active phase %) has not been investigated within the same islet. In this work, we show that low HCO₃ (5 mM) Hepes buffered solutions reversibly reduce the frequency of bursts compared to control (25 mM) HCO₃ buffered solutions in the same islet. There was no change in the threshold or active phase (%). Using the mathematical model of Sherman et al. 1988, we explored mechanisms for a change in frequency independent of a change in active phase (%). Increased exchangeable calcium pool size and increased cell to cell coupling were the two theoretical treatments which could reproduce the experimental data. We conclude that burst frequency can be modulated independent of the active phase and that alteration of a calcium pool size best fits the experimental data. Offprint requests to: P. B. Carroll     

The low molecular weight inhibitor of NCX1 interacts with a cytosolic domain that differs from the ion-transport site of the Na/Ca exchanger

The endogenous inhibitory factor (NCX(IF)) of the cardiac Na/Ca exchanger (NCX1) is a low molecular weight substance, which has a strong capacity to modulate the ventricle muscle contractility. Previously, we have shown that NCX(IF) can completely inhibit either the forward (Na(i)-dependent Ca-uptake) or reverse (Na(o)-dependent Ca-release) mode of Na/Ca exchange as well as its partial reaction, the Ca/Ca exchange. Although the preliminary studies have shown that NCX(IF) can rapidly (within few milliseconds) interact with a putative inhibitory site of the Na/Ca exchanger protein (or within its vicinity), it was not clear whether the NCX(IF) can directly interact with the ion transport sites of the exchanger protein or the interaction site of NCX(IF) is distinct from the ion-binding/transport site of NCX1. In order to segregate between these possibilities the NCX(IF) was tested for its capacity to compete with Ca at the cytosolic side by using the preparation of sarcolemma vesicles having predominantly the inside-out orientation. For this goal, the initial rates of Na(i)-dependent (45)Ca-uptake were measured in the presence of extravesicular (cytosolic) NCX(IF) under conditions in which the concentration of extravesicular Ca was varied (2-200 microM) and intravesicular Na was kept fixed at saturating concentration (160 mM). Under these conditions the NCX(IF) results in several fold decrease in V(max) values, while having no significant effect on the K(m). Taking into account the molecular weight of 350-550 Da (derived from the gel-filtration and mass-spectra data), the experimentally measured inhibitory potency of NCX(IF) can be estimated as the IC(50) = 0.3-0.6 microM. Therefore, it is concluded that the NCX(IF) is reasonably potent blocker, which interacts with cytosolic domain thereby preventing the ion-translocation (and not ion-binding) events.     

CO2 permeability of the rat erythrocyte membrane and its inhibition

We have studied the CO₂ permeability of the erythrocyte membrane of the rat using a mass spectrometric method that employs ¹⁸O-labelled CO₂. The method yields, in addition, the intraerythrocytic carbonic anhydrase activity and the membrane HCO₃⁻ permeability. For normal rat erythrocytes, we find at 37 °C a CO₂ permeability of 0.078 ± 0.015 cm/s, an intracellular carbonic anhydrase activity of 64,100, and a bicarbonate permeability of 2.1 × 10⁻³cm/s. We studied whether the rat erythrocyte membrane possesses protein CO₂ channels similar to the human red cell membrane by applying the potential CO₂ channel inhibitors pCMBS, Dibac, phloretin, and DIDS. Phloretin and DIDS were able to reduce the CO₂ permeability by up to 50%. Since these effects cannot be attributed to the lipid part of the membrane, we conclude that the rat erythrocyte membrane is equipped with protein CO₂ channels that are responsible for at least 50% of its CO₂ permeability.     

The distribution of carbonic anhydrase type I and II isozymes in lamprey and trout: possible co-evolution with erythrocyte chloride/bicarbonate exchange

The subcellular distribution and kinetic properties of carbonic anhydrase were examined in red blood cells and gills of the lamprey, Petromyzon marinus, a primitive agnathan, and rainbow trout, Oncorhynchus mykiss, a modern teleost, in relation to the evolution of rapid Cl^–/HCO ₃ ^– exchange in the membrane of red blood cells. In the lamprey, which either lacks or has minimal red cell Cl^–/HCO ₃ ^– exchange, there has been no compensatory incorporation of carbonic anhydrase into the membrane fraction of either the red cell or the gill. Carbonic anhydrase activity in red cells is exclusively cytoplasmic, and the single isozyme displays kinetic properties typical of the type I, slow turnover, isozyme. In the red blood cells of the trout, however, which possess high amounts of the band-3 Cl^–/HCO ₃ ^– exchange protein, the single carbonic anhydrase isozyme appears to be kinetically similar to the type II, fast turnover, isozyme. It thus appears that the type I isozyme present in the red blood cells of primitive aquatic vertebrates was replaced in modern teleosts by the kinetically more efficient type II isozyme only after the incorporation and expression of a significant amount of the band-3 exchange protein in the membrane of the red cell.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - CA carbonic anhydrase - DTT dithiothreitol - EDTA ethylenediaminetetra-acetate - E ₀ total concentration of free enzyme - i fractional inhibition of enzyme activity - IU international units - K ₁ inhibition constant - K _M Michaelis constant - NBT nitro blue tetrazolium - NCP nitrocellulose paper - RBC red blood cell - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max maximal velocity of reaction     

Effects of the internal recycling rate on biological nutrient removal and microbial community structure in a sequential anoxic/anaerobic membrane bioreactor

This study investigated the effects of the internal recycling rate on nutrients removal in a sequential anoxic/anaerobic membrane bioreactor (SAM). Microbial community structure in sludge from the SAM was studied using quinone profile method. Above 98% COD, 68% nitrogen, and 55% phosphorus removal efficiencies were achieved when the internal recycling rate was 2.5 times influent flow. At that rate, the optimum specific nitrate loading rate and COD/NO(3)-N ratio were found to be 2.24 mgNO(3)-N g(-1) MLSS h(-1) and 9.13, respectively. Batch tests demonstrated that anoxic condition suppressed phosphorus release, and that denitrification was also influenced by initial substrate concentration. Denitrification appeared to have some priority over phosphorus release for substrate uptake. Microbial community analysis revealed a predominance of the subclass beta-Proteobacteria. Furthermore, it was found that Rhodocyclus-related bacteria were efficient at phosphorus removal than Actinobacteria.     

Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion

In many cell types agonist-receptor activation leads to a rapid and transient release of Ca(2+) from intracellular stores via activation of inositol 1,4,5 trisphosphate (InsP(3)) receptors (InsP(3)Rs). Stimulated cells activate store- or receptor-operated calcium channels localized in the plasma membrane, allowing entry of extracellular calcium into the cytoplasm, and thus replenishment of intracellular calcium stores. Calcium entry must be finely regulated in order to prevent an excessive intracellular calcium increase. Junctate, an integral calcium binding protein of endo(sarco)plasmic reticulum membrane, (a) induces and/or stabilizes peripheral couplings between the ER and the plasma membrane, and (b) forms a supramolecular complex with the InsP(3)R and the canonical transient receptor potential protein (TRPC) 3 calcium entry channel. The full-length protein modulates both agonist-induced and store depletion-induced calcium entry, whereas its NH(2) terminus affects receptor-activated calcium entry. RNA interference to deplete cells of endogenous junctate, knocked down both agonist-activated calcium release from intracellular stores and calcium entry via TRPC3. These results demonstrate that junctate is a new protein involved in calcium homeostasis in eukaryotic cells.     

Substrate and inhibitor specificity of anion exchangers on the brush border membrane of rabbit ileum

Summary In previous studies we have found that several anions can be transported by an exchange process in rabbit ileal brush border membranes. We demonstrated exchanges of Cl for OH or HCO₃, SO₄ for OH, oxalate for OH, and oxalate for Cl. The purpose of these studies was to determine the number of distinct carriers mediating these exchanges. We utilized substrate and inhibitor specificity studies to distinguish between different anion exchange transporters. We conclude that SO₄OH and oxalate: OH exchange occur on the same carrier because: (i) pH-gradient stimulated transport of both¹⁴C-oxalate and³⁵SO₄ were equally sensitive tocis-inhibition by unlabeled SO₄ or oxalate; and (ii) both were inhibited equally by K. We conclude that oxalate: OH and oxalate: Cl exchanges occur on different carriers because: (i) Cl or SO₄ caused unequalcis-inhibition of these two exchanges; and (ii) as compared to oxalate: Cl exchange, oxalate: OH exchange was more sensitive to inhibition by probenecid and K and less sensitive to inhibition by bumetanide. Finally, we conclude that oxalate: Cl exchange and ClHCO₃ exchange occur on different carriers because: (i) ClHCO₃ exchange was almost completely insensitive tocis-inhibition by oxalate; and (ii) oxalate: Cl exchange was more sensitive to inhibition by DIDS and bumetanide than ClHCO₃ exchange. Thus we have found that there are at least three separate anion exchangers on rabbit ileal brush border: (i) a ClHCO₃ exchanger; (ii) a SO₄OH exchanger, which also transports oxalate; and (iii) an oxalate: Cl exchanger.     

Biotransformation of glycerol to 3-hydroxypropionaldehyde: Improved production by in situ complexation with bisulfite in a fed-batch mode and separation on anion exchanger

3-Hydroxypropionaldehyde (3HPA) is an important C3 chemical that can be produced from renewable glycerol by resting whole cells of Lactobacillus reuteri. However the process efficiency is limited due to substrate inhibition, product-mediated loss of enzyme activity and cell viability, and also formation of by-products. Complex formation of 3HPA with sodium bisulfite and subsequent binding to Amberlite IRA-400 was investigated as a means of in situ product recovery and for overcoming inhibition. The adsorption capacity and -isotherm of the resin were evaluated using the Langmuir model. The resin exhibited maximum capacity of 2.92 mmol complex/g when equilibrated with 45 mL solution containing an equilibrium mixture of 2.74 mmol 3HPA-bisulfite complex and 2.01 mmol free 3HPA. The dynamic binding capacity based on the breakthrough curve of 3HPA and its complex on passing a solution with 2.49 mmol complex and 1.65 mmol free 3HPA was 2.01 mmol/g resin. The bound 3HPA was desorbed from the resin using 0.20 M NaCl with a high purity as a mixture of complexed- and free 3HPA at a ratio of 0.77 mol/mol. Fed-batch biotransformation of glycerol (818.85 mmol) with in situ 3HPA complexation and separation on the bisulfite-functionalized resin resulted in an improved process with consumption of 481.36 mmol glycerol yielding 325.54 mmol 3HPA at a rate of 17.13 mmol/h and a yield of 68 mol%. Also, the cell activity was maintained for at least 28 h.     

Activation of the PI3K/Akt signaling pathway through P2Y2 receptors by extracellular ATP is involved in osteoblastic cell proliferation

We studied the PI3K/Akt signaling pathway modulation and its involvement in the stimulation of ROS 17/2.8 osteoblast-like cell proliferation by extracellular ATP. A dose- and time-dependent increase in Akt-Ser 473 phosphorylation (p-Akt) was observed. p-Akt was increased by ATPγS and UTP, but not by ADPβS. Akt activation was abolished by PI3K inhibitors and reduced by inhibitors of PI-PLC, Src, calmodulin (CaM) but not of CaMK. p-Akt was diminished by cell incubation in a Ca²⁺-free medium but not by the use of L-type calcium channel blockers. The rise in intracellular Ca²⁺ induced by ATP was potentiated in the presence of Ro318220, a PKC inhibitor, and attenuated by the TPA, a known activator of PKC. ATP-dependent p-Akt was diminished by TPA and augmented by Ro318220 treatment in a Ca²⁺-containing but not in a Ca²⁺-free medium. ATP stimulated the proliferation of both ROS 17/2.8 cells and rat osteoblasts through PI3K/Akt. In the primary osteoblasts, ATP induces alkaline phosphatase activity via PI3K, suggesting that the nucleotide promotes osteoblast differentiation. These results suggest that ATP stimulates osteoblast proliferation through PI-PLC linked-P2Y₂ receptors and PI3K/Akt pathway activation involving Ca²⁺, CaM and Src. PKC seems to regulate Akt activation through Src and the Ca²⁺ influx/CaM pathway.     

Depolarization of the membrane potential by beta-lactams as a signal to induce autolysis.

The effect of beta-lactam antibiotics that are known to inhibit cell wall biosynthesis and induce cell wall autolysis on the electrophysiological state of the plasma membrane in Streptomyces griseus was studied. Addition of various beta-lactam antibiotics induced a dose- and growth-stage-dependent depolarization of the membrane potential of Streptomyces griseus. The hydrolyzed biologically inactive derivative penicilloic acid had no depolarizing effect on the membrane potential. The ionophore gramicidin D, while depolarizing the membrane potential, also induced a dose-dependent increase in cell wall lysis. These observations suggest that alteration of the transmembrane potential could be an important signal in triggering cell wall autolysis of S. griseus.     

Cytochrome bd-I in Escherichia coli is less sensitive than cytochromes bd-II or bo′' to inhibition by the carbon monoxide-releasing molecule,CORM-3: N-acetylcysteine reduces CO-RM uptake and inhibition of respiration

Background: CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO – a critical gasotransmitter – in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident. Methods: Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli — cytochrome bd-I, cytochrome bd-II and cytochrome bo′, to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (K_m 0.24 μM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood. Results: We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo′. Cytochromes bo′ and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells. Conclusions: A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a potent inhibitor of CO-RM uptake by E. coli. General significance: Rational design and exploitation of CO-RMs require a fundamental understanding of their activity. CO and CO-RMs have multifaceted effects on mammalian and microbial cells; here we show that the quinol oxidases of E. coli are differentially sensitive to CORM-3. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Autophagy is used as a survival program in unfertilized sea urchin eggs that are destined to die by apoptosis after inactivation of MAPK1/3 (ERK2/1)

A high MAPK1/3 (also known as ERK2/1, respectively) activity, preventing spontaneous activation, is essential to maintain cell cycle arrest of mature oocytes of mammals, frogs or invertebrates such as starfish. Mature oocytes would undergo a “suicide”-like cell death if not fertilized. We previously have reported that downregulation of MAPK1/3 in unfertilized sea urchin eggs induces a calcium-dependent entry into mitosis. We show here that this event is followed by a series of pseudo-mitotic cell cycles associated with transient Ca_i increases, preceding CASP3/caspase-3 activation and apoptosis. However, cell death was delayed after inhibition of the Ca_i transients or of cyclin-dependent kinases (CDK), with roscovitine. In these conditions, eggs enter an autophagy program as suggested by detection of processed LC3B by western blot, immunofluorescence and immunogold staining, visualization of autophagy vesicles by electron microscopy, and an increase in acidic vesicular organelles (AVOs). We found that bafilomycin A₁ or an association of leupeptin and pepstatin, which are widely used to study autophagy, may act upon calcium signaling or cell cycle events, respectively, and not only on autophagy events. Finally, inhibition of PtdIns 3-kinase with wortmannin or LY294002 powerfully stimulated cell death of unfertilized eggs, which suggests that this activity does not negatively regulate autophagy as is often reported, but rather stimulates survival in unfertilized eggs. We suggest that apoptosis of unfertilized eggs is the consequence of an aberrant short attempt of development that occurs if MAPK1/3 is inactivated, but these eggs can use autophagy as a survival program when the cell cycle is blocked.     

End-products diacylglycerol and ceramide modulate membrane fusion induced by a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

A phospholipase C/sphingomyelinase from Pseudomonas aeruginosa has been assayed on vesicles containing phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and cholesterol at equimolar ratios. The enzyme activity modifies the bilayer chemical composition giving rise to diacylglycerol (DAG) and ceramide (Cer). Assays of enzyme activity, enzyme-induced aggregation and fusion have been performed. Ultrastructural evidence of vesicle fusion at various stages of the process is presented, based on cryo-EM observations. The two enzyme lipidic end-products, DAG and Cer, have opposite effects on the bilayer physical properties; the former abolishes lateral phase separation, while the latter generates a new gel phase [Sot et al., FEBS Lett. 582, 3230-3236 (2008)]. Addition of either DAG, or Cer, or both to the liposome mixture causes an increase in enzyme binding to the bilayers and a decrease in lag time of hydrolysis. These two lipids also have different effects on the enzyme activity, DAG enhancing enzyme-induced vesicle aggregation and fusion, Cer inhibiting the hydrolytic activity. These effects are explained in terms of the different physical properties of the two lipids. DAG increases bilayers fluidity and decreases lateral separation of lipids, thus increasing enzyme activity and substrate accessibility to the enzyme. Cer has the opposite effect mainly because of its tendency to sequester sphingomyelin, an enzyme substrate, into rigid domains, presumably less accessible to the enzyme.     

The novel chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA3-DTDA) promotes stable binding of His-tagged proteins to liposomal membranes: Potent anti-tumor responses induced by simultaneously targeting antigen, cytokine and costimulatory signals to T cells

Recent studies indicate that the chelator lipid nitrilotriacetic acid ditetradecylamine (NTA-DTDA) can be used to engraft T cell costimulatory molecules onto tumor cell membranes, potentially circumventing the need for genetic manipulation of the cells for development of cell- or membrane-based tumor vaccines. Here, we show that a related lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA₃-DTDA, which has three NTA moieties in its headgroup instead of one) is several-fold more effective than NTA-DTDA at promoting stable His-tagged protein engraftment. IAsys biosensor studies show that binding of His-tagged B7.1 (B7.1-6H) to NTA₃-DTDA-containing membranes, exhibit a faster on-rate and a slower off-rate, compared to membranes containing NTA-DTDA. Also, NTA₃-DTDA-containing liposomes and plasma membrane vesicles (PMV) engrafted with B7.1-6H and CD40-6H exhibit greater binding to T cells, in vitro and in vivo. Engrafted NTA₃-DTDA-containing PMV encapsulated cytokines such as IL-2, IL-12, GM-CSF and IFN-γ, allowing targeted delivery of both antigen and cytokine to T cells, and stimulation of antigen-specific T cell proliferation and cytotoxicity. Importantly, use of B7.1-CD40-engrafted PMV containing IL-2 and IL-12 as a vaccine in DBA/2J mice induced protection against challenge with syngeneic tumor cells (P815 mammary mastocytoma), and regression of established tumors. The results show that stable protein engraftment onto liposomal membranes using NTA₃-DTDA can be used to simultaneously target associated antigen, costimulatory molecules and cytokines to T cells in vivo, inducing strong anti-tumor responses and immunotherapeutic effect.     

Synergistic transmembrane insertion of the heterodimeric PGLa/magainin 2 complex studied by solid-state NMR

The skin secretions of amphibians are a rich source of antimicrobial peptides. The two antimicrobial peptides PGLa and magainin 2, isolated from the African frog Xenopus laevis, have been shown to act synergistically by permeabilizing the membranes of microorganisms. In this report, the literature on PGLa is extensively reviewed, with special focus on its synergistically enhanced activity in the presence of magainin 2. Our recent solid state ²H NMR studies of the orientation of PGLa in lipid membranes alone and in the presence of magainin 2 are described in detail, and some new data from 3,3,3-²H₃-L-alanine labeled PGLa are included in the analysis.