Extrusion Pathway Research Articles

Model for the allosteric regulation of the Na+/Ca2+ exchanger NCX

The Na(+) /Ca(2+) exchanger provides a major Ca(2+) extrusion pathway in excitable cells and plays a key role in the control of intracellular Ca(2+) concentrations. In Canis familiaris, Na(+) /Ca(2+) exchanger (NCX) activity is regulated by the binding of Ca(2+) to two cytosolic Ca(2+) -binding domains, CBD1 and CBD2, such that Ca(2+) -binding activates the exchanger. Despite its physiological importance, little is known about the exchanger's global structure, and the mechanism of allosteric Ca(2+) -regulation remains unclear. It was found previously that for NCX in the absence of Ca(2+) the two domains CBD1 and CBD2 of the cytosolic loop are flexibly linked, while after Ca(2+) -binding they adopt a rigid arrangement that is slightly tilted. A realistic model for the mechanism of the exchanger's allosteric regulation should not only address this property, but also it should explain the distinctive behavior of Drosophila melanogaster's sodium/calcium exchanger, CALX, for which Ca(2+) -binding to CBD1 inhibits Ca(2+) exchange. Here, NMR spin relaxation and residual dipolar couplings were used to show that Ca(2+) modulates CBD1 and CBD2 interdomain flexibility of CALX in an analogous way as for NCX. A mechanistic model for the allosteric Ca(2+) regulation of the Na(+) /Ca(2+) exchanger is proposed. In this model, the intracellular loop acts as an entropic spring whose strength is modulated by Ca(2+) -binding to CBD1 controlling ion transport across the plasma membrane.

Regulation of neuronal pH by the metabotropic Zn(2+)-sensing Gq-coupled receptor, mZnR/GPR39.

Synaptically released Zn(2+) acts as a neurotransmitter, in part, by activating the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39). In previous work using epithelial cells, we described crosstalk between Zn(2+) signaling and changes in intracellular pH and/or extracellular pH (pHe). As pH changes accompany neuronal activity under physiological and pathological conditions, we tested whether Zn(2+) signaling is involved in regulation of neuronal pH. Here, we report that up-regulation of a major H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), is induced by mZnR/GPR39 activation in an extracellular-regulated kinase 1/2-dependent manner in hippocampal neurons in vitro. We also observed that changes in pHe can modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. Similarly, Zn(2+)-dependent extracellular-regulated kinase 1/2 phosphorylation and up-regulation of NHE activity were absent at acidic pHe. Thus, our results suggest that when pHe is maintained within the physiological range, mZnR/GPR39 activation can up-regulate NHE-dependent recovery from intracellular acidification. During acidosis, as pHe drops, mZnR/GPR39-dependent NHE activation is inhibited, thereby attenuating further H(+) extrusion. This mechanism may serve to protect neurons from excessive decreases in pHe. Thus, mZnR/GPR39 signaling provides a homeostatic adaptive process for regulation of intracellular and extracellular pH changes in the brain. We show that the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39) activation induces up-regulation of a major neuronal H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), thereby enhancing neuronal recovery from intracellular acidification. Changes in extracellular pH (pHe), however, modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. This mechanism may serve to protect neurons from excessive decreases in pHe during acidosis. Hence, mZnR/GPR39 signaling provides a homeostatic adaptive process for regulation of intracellular and extracellular pH changes in the brain.

Septic cardiomyopathy in rat LPS-induced endotoxemia: relative contribution of cellular diastolic Ca(2+) removal pathways, myofibrillar biomechanics properties and action of the cardiotonic drug levosimendan.

Cardiac dysfunction is a common complication in sepsis and is characterized by forward pump failure. Hallmarks of septic cardiomyopathy are decreased myofibrillar contractility and reduced Ca(2+) sensitivity but it is still not clear whether reduced pump efficiency is predominantly a diastolic impairment. Moreover, a comprehensive picture of upstream Ca(2+) handling mechanisms and downstream myosin biomechanical parameters is still missing. Ca(2+)-sensitizing agents in sepsis may be promising but mechanistic insights for drugs like levosimendan are scarce. Here, we used an endotoxemic LPS rat model to study mechanisms of sepsis on in vivo hemodynamics, multicellular myofibrillar Ca(2+) sensitivity, in vitro cellular Ca(2+) homeostasis and subcellular actomyosin interaction with intracardiac catheters, force transducers, confocal Fluo-4 Ca(2+) recordings in paced cardiomyocytes, and in vitro motility assay, respectively. Left ventricular ejection fraction and myofibrillar Ca(2+) sensitivity were depressed in LPS animals but restored by levosimendan. Diastolic Ca(2+) transient kinetics was slowed down by LPS but ameliorated by levosimendan. Selectively blocking intracellular and sarcolemmal Ca(2+) extrusion pathways revealed minor contribution of sarcoplasmic reticulum Ca(2+) ATPase (SERCA) to Ca(2+) transient diastole in LPS-evoked sepsis but rather depressed Na(+)/Ca(2+) exchanger and plasmalemmal Ca(2+) ATPase. This was mostly compensated by levosimendan. Actin sliding velocities were depressed in myosin heart extracts from LPS rats. We conclude that endotoxemia specifically impairs sarcolemmal diastolic Ca(2+) extrusion pathways resulting in intracellular diastolic Ca(2+) overload. Levosimendan, apart from stabilizing Ca(2+)-troponin C complexes, potently improves cellular Ca(2+) extrusion in the septic heart.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Efflux by Small Multidrug Resistance Proteins Is Inhibited by Membrane-interactive Helix-stapled Peptides

Bacterial cell membranes contain several protein pumps that resist the toxic effects of drugs by efficiently extruding them. One family of these pumps, the small multidrug resistance proteins (SMRs), consists of proteins of about 110 residues that need to oligomerize to form a structural pathway for substrate extrusion. As such, SMR oligomerization sites should constitute viable targets for efflux inhibition, by disrupting protein-protein interactions between helical segments. To explore this proposition, we are using Hsmr, an SMR from Halobacter salinarum that dimerizes to extrude toxicants. Our previous work established that (i) Hsmr dimerization is mediated by a helix-helix interface in Hsmr transmembrane (TM) helix 4 (residues (90)GLALIVAGV(98)); and (ii) a peptide comprised of the full TM4(85-105) sequence inhibits Hsmr-mediated ethidium bromide efflux from bacterial cells. Here we define the minimal linear sequence for inhibitor activity (determined as TM4(88-100), and then "staple" this sequence via Grubbs metathesis to produce peptides typified by acetyl-A-(Sar)3-(88)VVGLXLIZXGVVV(100)-KKK-NH2 (X = 2-(4'-pentenyl)alanine at positions 92 and 96; Z = Val, Gly, or Asn at position 95)). The Asn(95) peptide displayed specific efflux inhibition and resensitization of Hsmr-expressing cells to ethidium bromide; and was non-hemolytic to human red blood cells. Stapling essentially prevented peptide degradation in blood plasma and liver homogenates versus an unstapled counterpart. The overall results confirm that the stapled analog of TM4(88-100) retains the structural complementarity required to disrupt the Hsmr TM4-TM4 locus in Hsmr, and portend the general validity of stapled peptides as therapeutics for the disruption of functional protein-protein interactions in membranes.

Fault Slip Rates and Seismic Moment Deficits on Major Active Faults in Middle and South Parts of the Eastern Margin of the Tibetan Plateau

AbstractAs the pathway for southeastward extrusion of the Tibetan Plateau, the middle and south parts of the eastern margin of the Tibetan Plateau are characterized by widespread active faults, and intensive and frequent seismic activity. In this paper, we use the spherical linear block theory constrained by geodetic observations from Global Positioning System (GPS) stations to estimate fault slip rates of active faults in this area. A 3D geometric block model is established based on previous relevant studies of active blocks. Using these received slip rates and optimal locking depths of faults, the rates of moment accumulation on the major faults are estimated. And then we compare them with the seismic moment released on each fault using the historical earthquake catalog. The results show that there are six faults or fault segments with large moment deficits in the research area, which would be helpful for research of middle‐long term earthquake hazard.

Reduced Antimony Accumulation in ARM58 -Overexpressing Leishmania infantum

Antimony-based drugs are still the mainstay of chemotherapy against Leishmania infections in many countries where the parasites are endemic. The efficacy of antimonials has been compromised by increasing numbers of resistant infections, the basis of which is not fully understood and likely involves multiple factors. By using a functional cloning strategy, we recently identified a novel antimony resistance marker, ARM58, from the parasite Leishmania braziliensis that protects the parasites against antimony-based antileishmanial compounds. Here we show that the Leishmania infantum homologue also confers resistance against antimony but not against other antileishmanial drugs and that its function depends critically on one of four conserved domains of unknown function. This critical domain requires at least two hydrophobic amino acids and is predicted to form a transmembrane structure. Overexpression of ARM58 in antimony-exposed parasites reduces the intracellular Sb accumulation by over 70%, indicating a role for ARM58 in Sb extrusion pathways, but without involvement of energy-dependent transporter proteins.

Purinergic stimulation of K+-dependent Na+/Ca2+ exchanger isoform 4 requires dual activation by PKC and CaMKII

K+-dependent Na+/Ca2+-exchanger isoform 4 (NCXK4) is one of the most broadly expressed members of the NCKX (K+-dependent Na+/Ca2+-exchanger) family. Recent data indicate that NCKX4 plays a critical role in controlling normal Ca2+ signal dynamics in olfactory and other neurons. Synaptic Ca2+ dynamics are modulated by purinergic regulation, mediated by ATP released from synaptic vesicles or from neighbouring glial cells. Previous studies have focused on modulation of Ca2+ entry pathways that initiate signalling. Here we have investigated purinergic regulation of NCKX4, a powerful extrusion pathway that assists in terminating Ca2+ signals. NCKX4 activity was stimulated by ATP through activation of the P2Y receptor signalling pathway. Stimulation required dual activation of PKC (protein kinase C) and CaMKII (Ca2+/calmodulin-dependent protein kinase II). Mutating T312, a putative PKC phosphorylation site on NCKX4, partially prevented purinergic stimulation. These data illustrate how purinergic regulation can shape the dynamics of Ca2+ signalling by activating a signal damping and termination pathway.

IMAGING PERIVASCULAR TRANSPORT IN THE BRAIN

Amyloid-beta is removed from the brain via active transport over the blood-brain barrier. In addition, a significant amount of amyloid-beta is suggested to be transported along the walls of arteries. This pathway could be affected in cardiovascular pathological conditions, because of changes in vascular stiffness or composition of the extracellular matrix. Eventually, this could lead to deposition of amyloid-beta in the blood vessel wall. In order to study the mechanisms of this extrusion pathway, we have developed a cranial window setup in which side-stream darkfield imaging is combined with fluorescence imaging. We injected FITC-dextran in the brain, at a dept of 3 mm. Within minutes after injection, fluorescence was observed around the arteries, but not the veins. Fluorescence was observed in a clearly confined space, in which occasionally particles were observed that appeared to float freely. After death, the perivascular space disappeared, and appeared to collapse around the artery. Injection of FITC-dextran in dead animals did not result in a perivascular fluorescent signal. In conclusion, a perivascular transport pathway is present along the wall of arteries in the brain. This pathway has a large capacity, suggesting an effective extrusion pathway for amyloid-beta and other molecules in the brain. Left panel: fluorescent image of a cerebral artery. Perivascular fluorescence is observed. Right panel: side-stream dark field image of the same location. Scale bar is 100 microns.

Mitochondrial Calcium Uptake: Context Matters

Mitochondrial calcium (Ca2+) fluxes regulate ATP generation, contribute to the regulation of apoptosis, and when very large can play a role in buffering changes in intracellular Ca2+ concentration ([Ca2+]i). However, fundamental disagreements on the extent and speed of Ca2+ uptake by mitochondria have been reported. The reliability and consistency mitochondrial Ca2+ uptake measurements is consequently critical for our understanding of cell biology and pathology in cells from all tissue including heart, neurons, and kidney. Importantly, measurement of Ca2+ movement across the inner mitochondrial membrane (IMM) has been dramatically enhanced by the molecular identification of the mitochondrial Ca2+ uniporter (MCU) and the mitochondrial Na+/Ca2+ exchanger (NCLX). First, we quantitatively analyze cardiac mitochondrial Ca2+ uptake experiments from the literature. We then interpret the results with respect to measurements conducted on recent MCU candidates which suggest that the conductance of a single MCU is ∼6-7 pS (in 105 mM [Ca2+]). Our quantitative analysis suggests three clear findings: 1. Under physiological conditions, Ca2+ influx into mitochondria is small and is dwarfed by other cytosolic Ca2+ extrusion pathways; 2. MCU-dependent Ca2+ uptake appears to be dependent on [Ca2+]i under physiological conditions; 3. There appear to be hundreds of MCU channels per mitochondrion but they are predominantly closed under physiological conditions. We conclude that under physiological conditions mitochondria do not act as a significant Ca2+ buffers in heart despite being capable of substantial Ca2+ accumulation if non-physiological conditions favor prolonged levels of high extra-mitochondrial [Ca2+].

K+-Cl− cotransporter-2 KCC2 in chicken cardiomyocytes

Using antibodies prepared against a unique region (exon 22-24) of rat K(+)-Cl(-) cotransporter-2 (KCC2), we confirmed that the ~140-kDa KCC2 protein is exclusively expressed in rat brain, but in chicken, we observed strong reactivity not only with the ~140-kDa KCC2 protein in brain but also a slightly larger ~145-kDa protein in heart. In silico analysis showed that while exon 22 of KCC2 is unique to this isoform in therian mammals, it is retained in KCC2's closest paralog, KCC4, of lower vertebrates, including chicken. To eliminate potential cross-reactivity with chicken KCC4, the antibodies were preadsorbed with blocking peptides prepared over the only two regions showing significant sequence identity to chicken KCC4. This completely eliminated antibody recognition of exogenously expressed chicken KCC4 but not of the ~145-kDa protein in chicken heart, indicating that chicken heart expresses KCC2. Real-time PCR confirmed robust KCC2 transcript expression in both chicken brain and heart. Chicken heart expressed predominantly the longer KCC2a splice variant consistent with the larger ~145-kDa protein in chicken heart. Immunofluorescence microscopy revealed prominent plasma membrane KCC2 labeling in chicken ventricular cardiomyocytes. We hypothesize that KCC2 is an important Cl(-) extrusion pathway in avian cardiomyocytes that counters channel-mediated Cl(-) loading during high heart rates with β-adrenergic stimulation. While KCC2 is absent from mammalian cardiomyocytes, understanding the role that the other KCC isoforms play in Cl(-) homeostasis of these cells represents a nascent area of research.

Modeling Dynamic Regulation of Mitochondrial free Ca2+: Effects of Ca2+ Sequestration and Precipitation

One of the most intriguing features of cardiac mitochondria is their ability to accumulate, retain and release enormous amount of Ca2+ via sophisticated Ca2+ transport and buffering systems. Mitochondrial free Ca2+ ([Ca2+]m) is mainly regulated by integrated function of Ca2+ uptake and extrusion pathways and Ca2+ sequestration and precipitation mechanisms. In spite of significant studies on the kinetics and regulation of mitochondrial Ca2+ transport systems, the mechanisms involved in mitochondrial Ca2+ sequestration and precipitation remain obscure. Here we developed a detailed computational model by integrating our recent biophysical models of cation transporters and a novel Ca2+ sequestration mechanism into our existing model of mitochondrial bioenergetics to quantitatively understand the dynamic regulation of [Ca2+]m. Experiments were conducted to measure time course of [Ca2+]m, [Ca2+]e, [NADH]m and pH in guinea pig heart mitochondria suspended in buffer media subjected to incremental concentrations of CaCl2 (0–40 μM) and NaCl (0–20 mM). Model analyses of the data suggest (1) with a constant Ca2+ buffering capacity, it is not possible to explain the dynamics of [Ca2+]m, (2) [Ca2+]m is largely influenced by a Ca2+‐dependent Ca2+ sequestration mechanism and least affected by Ca2+‐phosphate precipitation and, (3) there may be additional pathways for Ca2+ uptake (RaM), apart from the Ca2+ uniporter.

SARAF Inactivates the Store Operated Calcium Entry Machinery to Prevent Excess Calcium Refilling

Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca(2+) stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Usingheterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca(2+)-dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca(2+) signals and determining the content of the major intracellular Ca(2+) stores, a role that is likely to be important in protecting cells from Ca(2+) overfilling.

The role of Hv1 and CatSper channels in sperm activation

Elevations of sperm intracellular pH and Ca(2+) regulate sperm motility, chemotaxis, capacitation and the acrosome reaction, and play a vital role in the ability of the sperm cell to reach and fertilise the egg. In human spermatozoa, the flagellar voltage-gated proton channel Hv1 is the main H(+) extrusion pathway that controls sperm intracellular pH, and the pH-dependent flagellar Ca²(+) channel CatSper is the main pathway for Ca²(+) entry as measured by the whole-cell patch clamp technique. Hv1 and CatSper channels are co-localized within the principal piece of the sperm flagellum. Hv1 is dedicated to proton extrusion from flagellum and is activated by membrane depolarisation, an alkaline extracellular environment, the endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. The CatSper channel is strongly potentiated by intracellular alkalinisation. Since Hv1 and CatSper channels are located in the same subcellular domain, proton extrusion via Hv1 channels should induce intraflagellar alkalinisation and activate CatSper ion channels. Therefore the combined action of Hv1 and CatSper channels in human spermatozoa can induce elevation of both intracellular pH and Ca²(+) required for sperm activation in the female reproductive tract. Here, we discuss how Hv1 and CatSper channels regulate human sperm physiology and the differences in control of sperm intracellular pH and Ca²(+) between species.

Ca efflux via the sarcolemmal Ca ATPase occurs only in the t-tubules of rat ventricular myocytes

The transverse (t-) tubule network is an important site for Ca influx and release during excitation-contraction coupling in cardiac ventricular myocytes; however, its role in Ca extrusion is less clear. The present study was designed to investigate the relative contributions of Ca extrusion pathways across the t-tubule and surface membranes. Ventricular myocytes were isolated from the hearts of adult male Wistar rats and detubulated using formamide. Intracellular Ca was monitored using fluo-3 and confocal microscopy. Caffeine (20mmol/L) was used to induce SR Ca release; carboxyeosin (20μmol/L) and nickel (10mmol/L) were used to inhibit the sarcolemmal Ca ATPase and Na/Ca exchanger (NCX) respectively. Carboxyeosin decreased the rate constant of decay of the caffeine-induced Ca transient in control cells, but had no effect in detubulated cells, suggesting that Ca extrusion via the Ca ATPase occurs only across the t-tubule membrane. However nickel decreased the rate constant of the caffeine-induced Ca transient in control and detubulated cells, although its effect was greater in control cells, suggesting that Ca extrusion via NCX occurs across the surface and t-tubule membranes. The PKA inhibitor H-89 (10μmol/L) was used to investigate the role of basal PKA activity in Ca extrusion; H-89 appeared to have no effect on Ca extrusion via the Ca ATPase, but reduced Ca extrusion via NCX at the t-tubules but not the surface membrane. Thus it appears that Ca extrusion via the sarcolemmal Ca ATPase occurs only at the t-tubules, and is not regulated by basal PKA activity, while Ca extrusion via NCX occurs across both the surface and t-tubule membranes, but predominantly across the t-tubule membrane due, in part, to localised stimulation of NCX by PKA at the t-tubules. This may be important in heart disease, in which changes in t-tubule structure and protein phosphorylation occur.

Sodium-Calcium Exchangers in Rat Ameloblasts

Although the central role of ameloblasts in synthesis and resorption of enamel matrix proteins during amelogenesis is well documented, the Ca2+-transport/extrusion mechanism remains to be fully elucidated. To clarify Ca2+-transport in rat ameloblasts, we investigated expression and localization of Na+-Ca2+ exchanger (NCX) isoforms and the functional characteristics of their ion transporting/pharmacological properties. RT-PCR and immunohistochemical analyses revealed expression of NCX1 and NCX3 in ameloblasts, localized in the apical membrane. In patch-clamp recordings, Ca2+ efflux by Na+-Ca2+ exchange showed dependence on external Na+. Ca2+ influx by Na+-Ca2+ exchange, measured by fura-2 fluorescence, showed dependence on extracellular Ca2+ concentration, and it was blocked by NCX inhibitors KB-R7943, SEA0400, and SN-6. These results showed significant expression of NCX1 and NCX3 in ameloblasts, indicating their involvement in the directional Ca2+ extrusion pathway from cells to the enamel mineralizing front.

Functional Rotation of the Transporter AcrB: The Essentials of Peristaltic Motion and Subsequent Substrate Extrusion

Bacteria, such as E. coli, use multidrug efflux pumps to export toxic substrates through their cell membranes, including antibiotics. The RND transporter of the AcrAB-TolC efflux pump is able to export structurally and chemically different substrates via a functional rotation. The three major states of this rotation cycle were found in several asymmetric crystal structures. After initially analyzing the basic mechanisms of opening of the TolC channel [1] and of substrate extrusion by AcrB [2] separately, we have continued the analysis of the latter one. Thereby, we have focused both on the local interactions between substrate and protein, the properties of the extrusion pathway, as well as the principal subdomain movements which lead to the peristaltic motion. Furthermore, we have investigated the possibility to pull the substrate from the final state of the previous simulations out of the exit gate to estimate whether the substrate is already free to leave the protein via diffusion, which is usually beyond the time scale of computer simulations.[1] R. Schulz, U. Kleinekathofer, Biophys. J. 96, 3116 (2009)[2] R. Schulz, A. Vargiu, F. Collu, U. Kleinekathofer, P. Ruggerone, submitted

Encounter and extrusion of an intrahelical lesion by a DNA repair enzyme

How living systems detect the presence of genotoxic damage embedded in a million-fold excess of undamaged DNA is an unresolved question in biology. Here we have captured and structurally elucidated a base-excision DNA repair enzyme, MutM, at the stage of initial encounter with a damaged nucleobase, 8-oxoguanine (oxoG), nested within a DNA duplex. Three structures of intrahelical oxoG-encounter complexes are compared with sequence-matched structures containing a normal G base in place of an oxoG lesion. Although the protein-DNA interfaces in the matched complexes differ by only two atoms-those that distinguish oxoG from G-their pronounced structural differences indicate that MutM can detect a lesion in DNA even at the earliest stages of encounter. All-atom computer simulations show the pathway by which encounter of the enzyme with the lesion causes extrusion from the DNA duplex, and they elucidate the critical free energy difference between oxoG and G along the extrusion pathway.

788 Effect of Genetic Ablation of the Acid/Base Transporters PNBC1, NHE1, NHE2, and Slc26a7, On Gastric Epithelial Functions

Background: Recent investigations have revealed the expression of a multitude of basolateral acid/base transporters in gastric parietal cells, but the physiological significance of many of them is not established. Aim and methods: The study was designed to elucidate the physiological role of the Na/HCO3 cotransporter pNBC1, the Na+/H+ exchangers NHE1 and NHE2, all abundantly expressed in parietal cells. Isolated gastric mucosae from ten days old mice (as both NBC1and NHE1-deficient mice have a short life expectancy) were placed in classical Ussing chambers and basal and forskolin-stimulated acid secretory rate determined by back titration. Identical experiments were performed with selective pharmacological inhibitors of NBC and NHEs. Parietal cell ultrastructure was examined by electron microscopy, expression of H+/K+ATPase was assessed by quantitative RT-PCR and immunohistochemistry, epitheial cell apoptosis assessed by the TUNEL assay, and proliferation by immunohistochemistry with anti-Ki67, anti-histone. Results: Forskolin-stimulated acid secretion was significantly reduced (by >50%) in NBC1-deficient gastric mucosa, or after pharmacological inhibition of NBC1, but remained unaltered in NHE1or Slc26a7 deficient gastric mucosa in ten days old mice, or after pharmacological inhibition of NHE1 and NHE2. Parietal cell number, ultrastructure, H+/K+-ATPase expression and epithelial cell proliferation were normal in NBC1as well as NHE1 deficient mice. This indicates that pNBC1 likely serves as an additional base extrusion pathway in addition to the Cl/HCO3 exchanger AE2. In contrast, NHE2-deficient mucosae secreted acid only as a short burst, and displayed a reduction of H+/K+-ATPase but not AE2 expression. At postnatal day 10, no difference in apoptotic rate, proliferative indices, histology, and parietal cell numbers were detected in NHE2 +/+ and -/mice, whereas a complete change of the gastric proliferative zone and marked loss of parietal cell number was seen in adult stomach. Summary and conclusions: The electrogenic Na/HCO3 cotransporter NBC1 is an important base extruder during stimulation of acid secretion, and its absence or inhibition leads to a significant reduction in maximal acid secretory rates. In contrast, NHE1 and SLC26a7 anion transporter are not essential components of the ion transport machinery for gastric acid secretion in young mice. NHE2 ablation leaves parietal cell structure intact in young mice, but renders them non-functional.

Increased O2 consumption in excitation–contraction coupling in hypertrophied rat heart slices related to increased Na+–Ca2+ exchange activity

The goal of our study was to evaluate the origin of the increased O(2) consumption in electrically stimulated left ventricular slices of isoproterenol-induced hypertrophied rat hearts with normal left ventricular pressure. O(2) consumption per minute (mVO(2)) of mechanically unloaded left ventricular slices was measured in the absence and presence of 1-Hz field stimulation. Basal metabolic mVO(2), i.e., mVO(2) without electrical stimulation, was significantly smaller, but mVO(2) for the total Ca(2+) handling in excitation-contraction coupling (E-C coupling mVO(2)), i.e., delta mVO(2) (=mVO(2) with stimulation - mVO(2) without stimulation), was significantly larger in the hypertrophied heart. Furthermore, the fraction of E-C coupling mVO(2) was markedly altered in the hypertrophied heart. Namely, mVO(2) consumed by sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) was depressed by 40%; mVO(2) consumed by the Na(+)/K(+)-ATPase (NKA)-Na(+)/Ca(2+) exchange (NCX) coupling was increased by 100%. The depressed mVO(2) consumption by SERCA2 was supported by lower protein expressions of phosphorylated-Ser(16) phospholamban and SERCA2. The increase in NKA-NCX coupling mVO(2) was supported by marked augmentation of NCX current. However, the increase in NCX current was not due to the increase in NCX1 protein expression, but was attributable to attenuation of the intrinsic inactivation mechanisms. The present results demonstrated that the altered origin of the increased E-C coupling mVO(2) in hypertrophy was derived from decreased SERCA2 activity (1ATP: 2Ca(2+)) and increased NCX activity coupled to NKA activity (1ATP: Ca(2+)). Taken together, we conclude that the energetically less efficient Ca(2+) extrusion pathway evenly contributes to Ca(2+) handling in E-C coupling in the present hypertrophy model.