Intracellular Buffering Research Articles

K201 (JTV-519) alters the spatiotemporal properties of diastolic Ca2+ release and the associated diastolic contraction during β-adrenergic stimulation in rat ventricular cardiomyocytes

K201 has previously been shown to reduce diastolic contractions in vivo during β-adrenergic stimulation and elevated extracellular calcium concentration ([Ca2+]o). The present study characterised the effect of K201 on electrically stimulated and spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca2+ release and contractile events in isolated rat cardiomyocytes during β-adrenergic stimulation and elevated [Ca2+]o. Parallel experiments using confocal microscopy examined spontaneous diastolic Ca2+ release events at an enhanced spatiotemporal resolution. 1.0 μmol/L K201 in the presence of 150 nmol/L isoproterenol (ISO) and 4.75 mmol/L [Ca2+]o significantly decreased the amplitude of diastolic contractions to ~16% of control levels. The stimulated free Ca2+ transient amplitude was significantly reduced, but stimulated cell shortening was not significantly altered. When intracellular buffering was taken into account, K201 led to an increase in action potential-induced SR Ca2+ release. Myofilament sensitivity to Ca2+ was not changed by K201. Confocal microscopy revealed diastolic events composed of multiple Ca2+ waves (2–3) originating at various points along the cardiomyocyte length during each diastolic period. 1.0 μmol/L K201 significantly reduced the (a) frequency of diastolic events and (b) initiation points/diastolic interval in the remaining diastolic events to 61% and 71% of control levels respectively. 1.0 μmol/L K201 can reduce the probability of spontaneous diastolic Ca2+ release and their associated contractions which may limit the propensity for the contractile dysfunction observed in vivo.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-011-0218-4) contains supplementary material, which is available to authorized users.

Physiological effects of hypercapnia in the deep-sea bivalve Acesta excavata (Fabricius, 1779) (Bivalvia; Limidae)

The option of storing CO 2 in subsea rock formations to mitigate future increases in atmospheric CO 2 may induce problems for animals in the deep sea. In the present study the deep-sea bivalve Acesta excavata was subjected to environmental hypercapnia (pHSW 6.35, P CO 2 = 33,000 μatm) corresponding to conditions reported from natural CO 2 seeps. Effects on acid–base status and metabolic rate were related to time of exposure and subsequent recovery. During exposure there was an uncompensated drop in both hemolymph and intracellular pH. Intracellular pH returned to control values, while extracellular pH remained significantly lower during recovery. Intracellular non-bicarbonate buffering capacity of the posterior adductor muscle of hypercapnic animals was significantly lower than control values, but this was not the case for the remaining tissues analyzed. Oxygen consumption initially dropped by 60%, but then increased during the final stages of exposure, which may suggest a higher tolerance to hypercapnia than expected for a deep-living species.

Syntaxin-1A Interacts with Distinct Domains within Nucleotide-binding Folds of Sulfonylurea Receptor 1 to Inhibit β-Cell ATP-sensitive Potassium Channels

The ATP-sensitive potassium (K(ATP)) channel regulates pancreatic β-cell function by linking metabolic status to electrical activity. Syntaxin-1A (Syn-1A), a SNARE protein mediating exocytotic fusion, binds and inhibits the K(ATP) channel via the nucleotide-binding folds (NBFs) of its sulfonylurea receptor-1 (SUR1) regulatory subunit. In this study, we elucidated the precise regions within the NBFs required for Syn-1A-mediated K(ATP) inhibition, using in vitro binding assays, whole cell patch clamp and FRET assay. Specifically, NBF1 and NBF2 were each divided into three subregions, Walker A (W(A)), signature sequence linker, and Walker B (W(B)), to make GST fusion proteins. In vitro binding assays revealed that Syn-1A associates with W(A) and W(B) regions of both NBFs. Patch clamp recordings on INS-1 and primary rat β-cells showed that Syn-1A-mediated channel inhibition was reversed by co-addition of NBF1-W(B) (not NBF1-W(A)), NBF2-W(A), and NBF2-W(B). The findings were corroborated by FRET studies showing that these truncates disrupted Syn-1A interactions with full-length SUR1. To further identify the binding sites, series single-site mutations were made in the Walker motifs of the NBFs. Only NBF1-W(A) (K719M) or NBF2-W(A) (K1385M) mutant no longer bound to Syn-1A; K1385M failed to disrupt Syn-1A-mediated inhibition of K(ATP) channels. These data suggest that NBF1-W(A) (Lys-719) and NBF2-W(A) (Lys-1385) are critical for Syn-1A-K(ATP) channel interaction. Taken together, Syn-1A intimately and functionally associates with the SUR1-NBF1/2 dimer via direct interactions with W(A) motifs and sites adjacent to W(B) motifs of NBF1 and NBF2 but transduces its inhibitory actions on K(ATP) channel activity via some but not all of these NBF domains.

The Cystine/Cysteine Cycle and GSH Are Independent and Crucial Antioxidant Systems in Malignant Melanoma Cells and Represent Druggable Targets

Cancer chemoresistance is often due to upregulation of antioxidant systems. Therapeutic targeting of these systems is however hampered by their redundancy. Here, we have performed a functional dissection of the antioxidant systems in different melanoma cases aimed at the identification of the most effective redox active drug. We have identified two crucial antioxidant mechanisms: glutathione (GSH), the major intracellular redox buffer, and the cystine/cysteine cycle, which switches the extracellular redox state from an oxidized to a reduced state. The two mechanisms are independent in melanoma cells and may be substitutes for each other, but targeting both of them is lethal. Exposure to the pro-oxidant compound As(2)O(3) induces an antioxidant response. However, while in these cells the intracellular redox balance remains almost unaffected, a reduced environment is generated extracellularly. GSH depletion by buthioninesulfoximine (BSO), or cystine/cysteine cycle inhibition by (S)-4-carboxyphenylglycine (sCPG), enhanced the sensitivity to As(2)O(3). Remarkably, sCPG also prevented the remodeling of the microenvironment redox state. We propose that the definition of the prevalent antioxidant system(s) in tumors is crucial for the design of tailored therapies involving redox-directed drugs in association with pro-oxidant drugs. In melanoma cells, BSO is the best enhancer of As(2)O(3) sensitivity. However, since the strong remodeling of the microenvironmental redox state caused by As(2)O(3) may promote tumor progression, the concomitant use of cystine/cysteine cycle blockers is recommended.

In vitro inhibition of topoisomerase IIα by reduced glutathione.

In most cells, the major intracellular redox buffer is glutathione (GSH) and its disulfide-oxidized (GSSG) form. The GSH/GSSG system maintains the intracellular redox balance and the essential thiol status of proteins by thiol disulfide exchange. Topoisomerases are thiol proteins and are a target of thiol-reactive substances. In this study, the inhibitory effect of physiological concentration of GSH and GSSG on topoisomerase IIα activity in vitro was investigated. GSH (0-10 mM) inhibited topoisomerase IIα in a concentration-dependent manner while GSSG (1-100 µM) had no significant effect. These findings suggest that the GSH/GSSG system could have a potential in vivo role in regulating topoisomerase IIα activity.

In vitro profiling of epigenetic modifications underlying heavy metal toxicity of tungsten-alloy and its components

Tungsten-alloy has carcinogenic potential as demonstrated by cancer development in rats with intramuscular implanted tungsten-alloy pellets. This suggests a potential involvement of epigenetic events previously implicated as environmental triggers of cancer. Here, we tested metal induced cytotoxicity and epigenetic modifications including H3 acetylation, H3-Ser10 phosphorylation and H3-K4 trimethylation. We exposed human embryonic kidney (HEK293), human neuroepithelioma (SKNMC), and mouse myoblast (C2C12) cultures for 1-day and hippocampal primary neuronal cultures for 1-week to 50–200μg/ml of tungsten-alloy (91% tungsten/6% nickel/3% cobalt), tungsten, nickel, and cobalt. We also examined the potential role of intracellular calcium in metal mediated histone modifications by addition of calcium channel blockers/chelators to the metal solutions. Tungsten and its alloy showed cytotoxicity at concentrations >50μg/ml, while we found significant toxicity with cobalt and nickel for most tested concentrations. Diverse cell-specific toxic effects were observed, with C2C12 being relatively resistant to tungsten-alloy mediated toxic impact. Tungsten-alloy, but not tungsten, caused almost complete dephosphorylation of H3-Ser10 in C2C12 and hippocampal primary neuronal cultures with H3-hypoacetylation in C2C12. Dramatic H3-Ser10 dephosphorylation was found in all cobalt treated cultures with a decrease in H3 pan-acetylation in C2C12, SKNMC and HEK293. Trimethylation of H3-K4 was not affected. Both tungsten-alloy and cobalt mediated H3-Ser10 dephosphorylation were reversed with BAPTA-AM, highlighting the role of intracellular calcium, confirmed with 2-photon calcium imaging. In summary, our results for the first time reveal epigenetic modifications triggered by tungsten-alloy exposure in C2C12 and hippocampal primary neuronal cultures suggesting the underlying synergistic effects of tungsten, nickel and cobalt mediated by changes in intracellular calcium homeostasis and buffering.

A Reaction‐Diffusion Model of Acid‐Base Balance in a Xenopus Oocyte

We present a new three-dimensional mathematical model of a Xenopus oocyte. The model assumes that the oocyte is a perfectly symmetric spherical cell surrounded by the extracellular unconvected solution (EUS) which in turn is surrounded by the stirred bulk extracellular solution (BECS). The oocyte plasma membrane is assumed to be infinitely thin and permeable only to CO2. Using Fick's second law of diffusion and the law of mass action, diffusion and reaction processes are modeled in both EUS and intracellular fluid (ICF). The model accounts for the slow equilibration CO2 + H2O ⇄ H2CO3 as governed by the forward and reverse rate constants and for competing equilibria among the HCO3− and an indefinite number of non-HCO3− buffers. We present the results of numerical simulations when, in addition to the CO2/HCO3− buffer, a single mobile non-HCO3− buffer is present in the ICF and EUS. By solving the system of reaction-diffusion equations we analyze how the profiles of pH and solutes change in time and space as a result of the CO2 influx. Consistent with experimental data (Musa-Aziz et al, PNAS, 2009; ASN 2005); the model predicts that applying extracellular 1.5% CO2/10 mM HCO3− (fixed pH of 7.50) causes the familiar fall in pHi and a rise followed by decay in pHS. We also show how the presence of two non-HCO3− intracellular buffers with different mobilities and how the CO2 membrane permeability affect the predictions of the model.

Getting heart cells on the same wavelength: infrared triggering of Ca2+transients in cardiac myocytes

Getting heart cells on the same wavelength: infrared triggering of Ca²⁺transients in cardiac myocytes

Whole-Cell Electrical Activity Under Direct Mechanical Stimulus by AFM Cantilever Using Planar Patch Clamp Chip Approach

Patch clamp is a powerful tool for studying the properties of ion-channels and cellular membrane. In recent years, planar patch clamp chips have been fabricated from various materials including glass, quartz, silicon, silicon nitride, polydimethyl-siloxane (PDMS), and silicon dioxide. Planar patch clamps have made automation of patch clamp recordings possible. However, most planar patch clamp chips have limitations when used in combination with other techniques. Furthermore, the fabrication methods used are often expensive and require specialized equipments. An improved design as well as fabrication and characterization of a silicon-based planar patch clamp chip are described in this report. Fabrication involves true batch fabrication processes that can be performed in most common microfabrication facilities using well established MEMS techniques. Our planar patch clamp chips can form giga-ohm seals with the cell plasma membrane with success rate comparable to existing patch clamp techniques. The chip permits whole-cell voltage clamp recordings on variety of cell types including Chinese Hamster Ovary (CHO) cells and pheochromocytoma (PC12) cells, for times longer than most available patch clamp chips. When combined with a custom microfluidics chamber, we demonstrate that it is possible to perfuse the extra-cellular as well as intra-cellular buffers. The chamber design allows integration of planar patch clamp with atomic force microscope (AFM). Using our planar patch clamp chip and microfluidics chamber, we have recorded whole-cell mechanosensitive (MS) currents produced by directly stimulating human keratinocyte (HaCaT) cells using an AFM cantilever. Our results reveal the spatial distribution of MS ion channels and temporal details of the responses from MS channels. The results show that planar patch clamp chips have great potential for multi-parametric high throughput studies of ion channel proteins.

Editorial [Hot topic: Crucial Role of Redox Signaling in the Regulation of Heart Health (Guest Editor: Dipak K. Das)

Editorial [Hot topic: Crucial Role of Redox Signaling in the Regulation of Heart Health (Guest Editor: Dipak K. Das)

Gastrointestinal Factors Influencing Zinc Absorption and Homeostasis

Diet-derived luminal factors have a major influence on zinc available for uptake across the apical membrane of enterocytes. Malabsorption and possibly intestinal microbiota limit this zinc availability. The transporter ZIP4 is expressed along the entire gastrointestinal tract and acts as a major processor of dietary zinc for loading into enterocytes from the apical membrane. Zip4 and other Zip family genes expressed in the gastrointestinal tract are up-regulated in periods of dietary zinc restriction. This provides for powerful homeostatic control. The transporter ZIP14 is up-regulated along the entire gastrointestinal tract by proinflammatory conditions. Intracellular transporters such as ZnT7, influence the transcellular movement of zinc across the enterocyte. Metallothionein, an intracellular metal buffer, and the transporter ZnT1 at the basolateral membrane, regulate the amount of zinc released to the portal circulation for systemic distribution. Pancreatic release of zinc by acinar cells is through the secretory process and apical membrane and involves transporters ZnT2 and ZnT1, respectively. Expression of both transporters is zinc-responsive. Enterocytes and acinar cells constitutively express Zip5 at the basolateral membrane, where it may serve as a monitor of zinc status.

Effects of acute dilutional hyponatremia on acid-base changes and electrolyte concentrations in rats with bilateral renal pedicle ligation

To describe the effects of increasing the extracellular fluid (ECF) volume by approximately 20% on acid-base changes and electrolyte concentrations in anesthetized rats. 18 adult male Sprague-Dawley rats. Rats were assigned to a control group (n = 6 rats) and a treatment group (12). All rats were anesthetized, and instrumentation and bilateral renal pedicle ligation were performed. The treatment group was infused IV with sterile water throughout a 30-minute period. Acid-base variables and concentrations of electrolytes, lactate, albumin, phosphorus, and hemoglobin were measured before (baseline) and 30 and 60 minutes after onset of infusion. Anion gap, strong ion difference, strong ion gap, and contributions of sodium, chloride, albumin, phosphorus, and lactate concentrations to base excess were calculated at each time point. Infusion of sterile water led to an increase in ECF volume of approximately 18%. This had no effect on acid-base balance, compared with that in control rats. Infusion of sterile water caused a significant decrease in sodium, chloride, ionized calcium, lactate, and albumin concentrations, compared with concentrations in the control group. Anion gap and calculated effects of sodium, chloride, albumin, and lactate concentrations on base excess at 60 minutes differed significantly between infused and control rats. Infusion of sterile water did not cause clinically relevant dilutional acidosis. The acidotic impact of water administration was offset by generation of new bicarbonate via carbonic acid equilibration and intracellular buffering in combination with the alkalotic effects of decreases in albumin, phosphorus, and lactate concentrations.

Na+-dependent HCO3− Import by the slc4a10 Gene Product Involves Cl− Export

The slc4a10 gene encodes an electroneutral Na(+)-dependent HCO(3)(-) importer for which the precise mode of action remains unsettled. To resolve this issue, intracellular pH (pH(i)) recordings were performed upon acidification in the presence of CO(2)/HCO(3)(-) by 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorometry of stably slc4a10-transfected NIH-3T3 fibroblasts. slc4a10 expression induced a significant Na(+)-dependent pH(i) recovery, which was accompanied by an increase in the intracellular Na(+) concentration evaluated by use of the Na(+)-sensitive fluorophore CoroNa Green. The estimated Na(+):HCO(3)(-) stoichiometry was 1:2. Cl(-) is most likely the counterion maintaining electroneutrality because (i) Na(+)-dependent pH(i) recovery was eliminated in Cl(-)-depleted cells; (ii) acute extracellular Cl(-) removal led to a larger alkalization in slc4a10-transfected cells than in control cells; and (iii) the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive and Na(+)- and HCO(3)(-)-dependent (36)Cl(-)-efflux during pH(i) recovery was significantly greater in acidified slc4a10-transfected cells than in control cells. Charged amino acids specific to slc4a gene family members that transport Na(+) and are expected to move more HCO(3)(-) molecules/turnover were targeted by site-directed mutagenesis. Na(+)-dependent pH(i) recovery was reduced in each of the single amino acid mutated cell lines (E890A, E892A, H976L, and H980G) compared with wild type slc4a10-transfected cells and completely eliminated in quadruple mutant cells. In conclusion, the data suggest that slc4a10 expressed in mammalian cells encodes a Na(+)-dependent Cl(-)/HCO(3)(-) exchanger in which four specific charged amino acids seem necessary for ion transport.

An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite, Plasmodium falciparum

The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study, a combination of fluorescent ion indicators and (36)Cl(-) flux measurements was used to investigate the transport of Cl(-) and the Cl(-)-dependent transport of "H(+)-equivalents" in mature (trophozoite stage) parasites, isolated from their host erythrocytes. Removal of extracellular Cl(-), resulting in an outward [Cl(-)] gradient, gave rise to a cytosolic alkalinization (i.e. a net efflux of H(+)-equivalents). This was reversed on restoration of extracellular Cl(-). The flux of H(+)-equivalents was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and, when measured in ATP-depleted parasites, showed a pronounced dependence on the pH of the parasite cytosol; the flux was low at cytosolic pH values < 7.2 but increased steeply with cytosolic pH at values > 7.2. (36)Cl(-) influx measurements revealed the presence of a Cl(-) uptake mechanism with characteristics similar to those of the Cl(-)-dependent H(+)-equivalent flux. The intracellular concentration of Cl(-) in the parasite was estimated to be approximately 48 mm in situ. The data are consistent with the intraerythrocytic parasite having in its plasma membrane a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive transporter that, under physiological conditions, imports Cl(-) together with H(+)-equivalents, resulting in an intracellular Cl(-) concentration well above that which would occur if Cl(-) ions were distributed passively in accordance with the parasite's large, inwardly negative membrane potential.

Microfluidic cell arrays for metabolic monitoring of stimulated cardiomyocytes

An array of PDMS microchambers was aligned to an array of sensor electrodes and stimulating microelectrodes, which was used for the electrochemical monitoring of the metabolic activity of single isolated adult ventricular myocytes inside the chamber array, stimulated within a transient electric field. The effect of the accumulation of metabolic byproducts in the limited extracellular volume of the picolitre chambers was demonstrated by measuring single muscle cell contraction optically, while concomitant changes in intracellular calcium transients and pH were recorded independently using fluorescent indicator dyes. Both the amplitude of the cell shortening and the magnitude of the intracellular calcium transients decreased over time and both nearly ceased after 20 min of continuous stimulation in the limited extracellullar volume. The intracellular pH decreased gradually during 20 min of continuous stimulation after which a dramatic pH drop was observed, indicating the breakdown of the intracellular buffering capacity. After continuous stimulation, intracellular lactate was released into the microchamber through cell electroporation and was detected electrochemically at a lactate microbiosensor, within the chamber. A mitochondrial uncoupler was used to mimic ischaemia and thus to enhance the cellular content of lactate. Under these circumstances, intracellular lactate concentrations were found to have risen to approximately 15 mM. This array system has the potential of simultaneous electrochemical and optical monitoring of extracellular and intracellular metabolites from single beating heart cells at a controlled metabolic state.

The role of calcium in modulation of the kinetics of synchronous and asynchronous quantal release at the neuromuscular junction

To elucidate the mechanisms of calcium regulation of the kinetics of the evoked neurotransmitter quantal release, we have investigated the temporal parameters of acetylcholine secretion in the mouse neuro-muscular junction at varying extracellular calcium concentration, in the presence of calcium channel blockers or intracellular calcium buffers. Acetylcholine secretion was induced by the motor nerve stimulation at a low frequency, which did not produce facilitation of the neurotransmitter release. The analysis of histograms of synaptic delays of uniquantal endplate currents recorded during 50 ms after the presynaptic action potential revealed three components of the secretion process: early and late periods of synchronous release and a delayed asynchronous release. At reduced extracellular calcium level, the relative number of quanta released during the asynchronous phase of secretion increased, while the rate of quantal release during the early synchronous period decreased. The findings support the hypothesis of participation of low- and high-affinity calcium sensors with different calcium binding kinetics in regulation of, respectively, synchronous and asynchronous release of neurotransmitter quanta.

Aging, proteotoxicity, mitochondria, glycation, NAD+ and carnosine: possible inter-relationships and resolution of the oxygen paradox

It is suggested that NAD+ availability strongly affects cellular aging and organism lifespan: low NAD+ availability increases intracellular levels of glycolytic triose phosphates (glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate) which, if not further metabolized, decompose spontaneously into methylglyoxal (MG), a glycating agent and source of protein and mitochondrial dysfunction and reactive oxygen species (ROS). MG-damaged proteins and other aberrant polypeptides can induce ROS generation, promote mitochondrial dysfunction and inhibit proteasomal activity. Upregulation of mitogenesis and mitochondrial activity by increased aerobic exercise, or dietary manipulation, helps to maintain NAD+availability and thereby decreases MG-induced proteotoxicity. These proposals can explain the apparent paradox whereby aging is seemingly caused by increased ROS-mediated macromolecular damage but is ameliorated by increased aerobic activity. It is also suggested that increasing mitochondrial activity decreases ROS generation, while excess numbers of inactive mitochondria are deleterious due to increased ROS generation. The muscle- and brain-associated dipeptide, carnosine, is an intracellular buffer which can delay senescence in cultured human fibroblasts and delay aging in senescence-accelerated mice. Carnosine's ability to react with MG and possibly other deleterious carbonyl compounds, and scavenge various ROS, may account for its protective ability towards ischemia and ageing.

Remodelling Ca2+ Responsiveness of Cav2.3 by Cavb Subunits: Role of an N-Terminal Polyacidic Motif

Ca2+-dependent inactivation of Cav2 channels is highly sensitive to intracellular Ca2+ buffers. Therefore, it seems likely that the cytoplasmic Ca2+ buffering scenario will have a large impact on the activity of Cav2.3 channels, which mediate Ca2+ influx associated with medium to slow neurotransmitter release. Using the whole-cell patch-clamp technique, here we show that the kinetics of the fast and slow components of macroscopic inactivation, τf and τs, of Cav2.3 are significantly slower when the cell is dyalized with 0.5 mM EGTA than when is dyalized with a solution containing no intracellular chelators. Rat Cavβ3 and a Cavβ subunit from the human parasite Schistosoma mansoni (CaβSm) eliminate the sensitivity of τf, but not of τs, to 0.5 mM EGTA. Interestingly, CavβSmalso eliminates the sensitivity of τf to 5 mM BAPTA, whereas Cavβ3 does not. Differently from mammalian Cavβ's, CavβSm contains a long N-terminal poly-acidic motif (NPAM). Does this motif interfere with responsiveness of τf to BAPTA? Coexpression with a CavβSm subunit without NPAM increased the sensitivity of τf to 5 mM BAPTA and enhanced the sensitivity of τs to EGTA and BAPTA. Coexpression with a chimaeric Cavβ3 subunit that contains an NPAM suppressed the sensitivity of both τf and τs to intracellular buffering. Thus, we conclude that presence of NPAM in Cavβ subunits reduces or suppresses the sensitivity of Cav2.3 inactivation to intracellular chelators. Perhaps NPAMs compete for Ca2+ with cellular buffers in the microdomains associated with Cav channels. We propose that the NPAM is a built-in buffer within the architecture of the CavβSm subunit with a function in modulating inactivation of schistosome Cav channels. Recombinant mammalian Cavβ subunits containing NPAMs could potentially offer a novel therapeutic strategy for diseases associated with enhanced Ca2+ entry.

Effect of beta-alanine supplementation on muscle carnosine concentrations and exercise performance

High-intensity exercise results in reduced substrate levels and accumulation of metabolites in the skeletal muscle. The accumulation of these metabolites (e.g. ADP, Pi and H(+)) can have deleterious effects on skeletal muscle function and force generation, thus contributing to fatigue. Clearly this is a challenge to sport and exercise performance and, as such, any intervention capable of reducing the negative impact of these metabolites would be of use. Carnosine (beta-alanyl-L-histidine) is a cytoplasmic dipeptide found in high concentrations in the skeletal muscle of both vertebrates and non-vertebrates and is formed by bonding histidine and beta-alanine in a reaction catalysed by carnosine synthase. Due to the pKa of its imidazole ring (6.83) and its location within skeletal muscle, carnosine has a key role to play in intracellular pH buffering over the physiological pH range, although other physiological roles for carnosine have also been suggested. The concentration of histidine in muscle and plasma is high relative to its K (m) with muscle carnosine synthase, whereas beta-alanine exists in low concentration in muscle and has a higher K (m) with muscle carnosine synthase, which indicates that it is the availability of beta-alanine that is limiting to the synthesis of carnosine in skeletal muscle. Thus, the elevation of muscle carnosine concentrations through the dietary intake of carnosine, or chemically related dipeptides that release beta-alanine on absorption, or supplementation with beta-alanine directly could provide a method of increasing intracellular buffering capacity during exercise, which could provide a means of increasing high-intensity exercise capacity and performance. This paper reviews the available evidence relating to the effects of beta-alanine supplementation on muscle carnosine synthesis and the subsequent effects on exercise performance. In addition, the effects of training, with or without beta-alanine supplementation, on muscle carnosine concentrations are also reviewed.

Analogues of the Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Antagonist Ned-19 Indicate Two Binding Sites on the NAADP Receptor

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+)-releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca(2+) release and NAADP binding. A fluorometry bioassay was used to assess NAADP-mediated Ca(2+) release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca(2+) release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca(2+) release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high- and one low-affinity) on the NAADP receptor.