Effects of sodium on chemotactic peptide binding to polymorphonuclear leukocytes

The affinity of binding of the chemotactic peptide N-formylnorleucylleucylphenylalanine to rabbit peritoneal polymorphonuclear leukocytes is increased when sodium ions are removed from the medium. In Hanks' balanced salt solution, the dissociation constant of the binding is about 2 X 10(-8) M, while in Na+-free medium, the dissociation constant is between 3 and 6 X 10(-9) M. Removal of Na+ appears to cause little or no change in receptor number. The change in affinity is rapid and reversible, occurs at 4 degrees C as well as 37 degrees C, and occurs when the Na+ is replaced by K+, choline, or sucrose. The increased binding of low concentrations of peptide is seen on broken as well as whole cells and therefore does not depend on an ion gradient across the membrane. The high affinity receptors are functional in mediating peptide uptake and lysosomal enzyme release. The receptors undergo down-regulation in Na+-free medium, and the dose dependence of the receptor loss is shifted to lower concentrations consistent with the higher affinity of the binding.     

Deficiency of joining of Okazaki-type fragments in absence of cellular protein synthesis.

The dependence of integration of newly formed DNA chain ( less than 10 S) into larger DNA on concomitant protein synthesis was studied in a special cellular system. Exponentially growing Ehrlich ascites tumor cells in vivo show decreasing rated and finally complete cessation of protein and DNA synthesis upon transfer into an isotonic but non-nutritive environment (Hanks' balanced salt solution). Both protein and DNA synthesis is stimulated in these cells for a period of 30 min when they are placed into fresh Hanks' balanced salt solution; however, stimulation of protein synthesis is completely prevented in Hanks' balanced salt solution containing cycloheximide. This system allowed us to investigate the formation and fate of newly formed DNA chains ( less than 10 S) in dependence of protein synthesis. Analysis of DNA produced in [3H]thymidine pulses showed that DNA chains smaller than 18 S were still formed during the phase of totally delayed protein synthesis and in the presence of cycloheximide, but they were not converted into DNA molecules sedimenting faster than 18 S under these conditions. Stimulation of protein synthesis for a period of 30 min allowed the short DNA pieces to be chased into larger DNA 30 min post stimulation of protein synthesis. The results clearly indicate that DNA chain growth, by sealing of DNA chains smaller than 18 S, is strongly dependent of concomitant protein synthesis. Direct chain elongation by addition of new deoxyribonucleotides is less dependent on concomitant cellular protein synthesis.     

Membrane attachment of Slr0006 in Synechocystis sp. PCC 6803 is determined by divalent ions

Slr0006 is one of the Synechocystis sp. PCC 6803 proteins strongly induced under carbon limiting conditions. Slr0006 has no predicted transmembrane helices or signal peptide sequence, yet it was exclusively recovered in the membrane fraction of Synechocystis, when the cells were broken in isolation buffers which contain divalent cations and are generally used for photosynthesis studies. Even subsequent washing of the membranes with high salt or various detergents did not release Slr0006, indicating strong binding of the Slr0006 protein to the membranes. Further, DNAse or RNAse treatment did not disturb the tight binding of Slr0006 protein to the membranes. Nevertheless, when the cells were broken in the absence of divalent cations, Slr0006 remained completely soluble. Binding of the Slr0006 to the membrane could not be properly reconstituted if the cations were added after breaking the cells in the absence of divalent ions. This unusual phenomenon has to be considered in identification and localization of other yet uncharacterized cyanobacterial proteins.     

Glutamine is a powerful effector of heat shock protein expression in Drosophila Kc cells.

We have investigated the effects of extracellular anions on the regulation of expression of the heat shock response in Drosophila Kc cells incubated in defined balanced salt solutions. Widely varying chloride concentrations had no effect on normal or heat shock protein (hsp) expression. Increasing glutamate concentrations from zero to 15 mM increased hsp expression more than 100-fold while affecting expression of non-heat-shock proteins minimally. Glutamine was 20-100-fold more potent than glutamate in supporting hsp expression, while other amino acids were less effective or supported no detectable hsp synthesis in heat shock. Inhibition of glutamine synthetase with methionine-sulfoximine resulted in very low hsp expression with glutamate and normal high level expression with glutamine, confirming the importance of glutamine. The absence of glucose and treatment with 2-deoxyglucose did not change the requirement for adequate glutamine for hsp expression. Cells heat shocked under conditions which gave very low hsp expression resumed growth when returned to normal medium as well as cells which expressed normal levels of hsps. Measurements of free amino acid levels in cells heat shocked in the presence and absence of glutamine showed a correlation between glutamine levels and amount of hsp expression. We conclude that a physiological process regulated by glutamine or a glutamine metabolite is important for normal hsp expression in heat shock conditions in Drosophila.     

Distribution and Ontogeny of 1S,3R-1-Aminocyclopentane-1,3-Dicarboxylic Acid-Sensitive and Quisqualate-Insensitive [3H]Glutamate Binding Sites in the Rat Brain

Abstract: Displacement of [³H]glutamate by 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid and quisqualate (in the presence of saturating concentrations of ionotropic glutamate receptor agonists) was used to characterize optimal ionic conditions, distribution, and the ontogeny of glutamate receptor binding sites in rat brain. Using rat forebrain membranes or receptor autoradiography, optimal 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid-sensitive [³H]glutamate binding was found in the presence of 100 m M bromide ions and in the absence of calcium ions. Under these conditions, [³H]glutamate binding was relatively quisqualate insensitive. In regions of the neonatal (11-day-old) and adult rat brain, this [³H]glutamate binding was highest in forebrain (striatum, cerebral cortex, and hippocampus) and hypothalamus/midbrain but was lower in the cerebellum, olfactory bulb, and pons/medulla regions. 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid-sensitive and quisqualate-insensitive [³H]glutamate binding was present in the rat forebrain at 1 day of age and gradually increased more than twofold by day 50 (adult). Thus, in the presence of bromide ions and in the absence of calcium ions, [³H]glutamate labels a subpopulation of metabotropic glutamate receptors that are sensitive to 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid but insensitive to quisqualate. Expression of [³H]glutamate binding under these conditions was both regionally and developmentally regulated in rat brain, suggesting that [³H]glutamate is labeling a distinct population of metabotropic glutamate receptors.     

Mg2+ is not catalytically required in the intrinsic and kirromycin-stimulated GTPase action of Thermus thermophilus EF-Tu

The influence of divalent metal ions on the intrinsic and kirromycin-stimulated GTPase activity in the absence of programmed ribosomes and on nucleotide binding affinity of elongation factor Tu (EF-Tu) from Thermus thermophilus prepared as the nucleotide- and Mg(2+)-free protein has been investigated. The intrinsic GTPase activity under single turnover conditions varied according to the series: Mn(2+) (0.069 min(-1)) > Mg(2+) (0.037 min(-1)) approximately no Me(2+) (0.034 min(-1)) > VO(2+) (0.014 min(-1)). The kirromycin-stimulated activity showed a parallel variation. Under multiple turnover conditions (GTP/EF-Tu ratio of 10:1), Mg(2+) retarded the rate of hydrolysis in comparison to that in the absence of divalent metal ions, an effect ascribed to kinetics of nucleotide exchange. In the absence of added divalent metal ions, GDP and GTP were bound with equal affinity (K(d) approximately 10(-7) m). In the presence of added divalent metal ions, GDP affinity increased by up to two orders of magnitude according to the series: no Me(2+) < VO(2+) < Mn(2+) approximately Mg(2+) whereas the binding affinity of GTP increased by one order of magnitude: no Me(2+) < Mg(2+) < VO(2+) < Mn(2+). Estimates of equilibrium (dissociation) binding constants for GDP and GTP by EF-Tu on the basis of Scatchard plot analysis, together with thermodynamic data for hydrolysis of triphosphate nucleotides (Phillips, R. C., George, P., and Rutman, R. J. (1969) J. Biol. Chem. 244, 3330-3342), showed that divalent metal ions stabilize the EF-Tu.Me(2+).GDP complex over the protein-free Me(2+).GDP complex in solution, with the effect greatest in the presence of Mg(2+) by approximately 10 kJ/mol. These combined results show that Mg(2+) is not a catalytically obligatory cofactor in intrinsic and kirromycin-stimulated GTPase action of EF-Tu in the absence of programmed ribosomes, which highlights the differential role of Mg(2+) in EF-Tu function.     

Caffeine sensitization of Chinese hamster ovary cells to heat killing

The effects of the methylxanthine, caffeine, on heat sensitization was investigated using Chinese hamster ovary (CHO) cells. Caffeine sensitized CHO cells to heat killing by reducing both the shoulder and the slope of the 44 degrees C survival curve. Heating was performed in suspension by addition of cells to preheated spinner flasks containing caffeine. Changes in intracellular free calcium levels, [Ca2+]i, were measured at 37 degrees C using the luminescent probe aequorin. Caffeine (1-5 mM) induced a transient increase in [Ca2+]i at 37 degrees C. The transient increase in [Ca2+]i was reduced 15-fold when 5 mM caffeine was added to aequorin-loaded cells suspended in Ca(2+)-free Hanks' balanced salt solution. However, 5 mM caffeine sensitized the cells to the same extent when they were suspended in either Ca(2+)-containing or Ca(2+)-free Hanks' balanced salt solution. The mechanism of heat sensitization by caffeine is still unknown.     

Chromatographic separations of serum proteins on immobilized metal ion stationary phases

The separation of proteins on stationary phases consisting of a bound organic chelator and a chelated divalent transition metal has been studied as a function of (A) metal ion species; (B) mobile phase composition and pH; and (C) anion and cation concentration. Optimum separation was observed at alkaline pH on chelated nickel stationary phases. Ammonium and Tris salts reduced the affinity of the metal chelate packing for serum proteins. Halide ions caused the proteins to be more strongly bound to the stationary phase. High salt concentrations had only a small effect on the binding of serum proteins in the absence of amine containing buffers or salts. It was also observed that the ease of elution and the recovery of protein were dependent on pH and upon the presence of halides. The general order of elution of serum proteins, based on isoelectric focusing, was independent of metal ion species and elution conditions, suggesting that a single mechanism or a unique sequence of mechanisms was operative. The results suggest that ligand exchange is the major mechanism of separation under basic conditions and that hydrophobic effects are the result of the competition of nonnitrogen ions with ammonium ions or amines for ligand binding sites modifying or participating in protein binding. Protein binding studies under weak acidic conditions are also presented although the mechanism responsible for protein binding is unclear.     

Chemical and physical factors affecting the excystation of Cryptosporidium parvum oocysts.

Cryptosporidium parvum oocysts were examined to ascertain excystation requirements and the effects of gamma irradiation. Oocysts and excysted sporozoites were examined for dye permeability and infectivity. Maximum excystation occurred when oocysts were pretreated with acid and incubated with bile salts, and potassium or sodium bicarbonate. Pretreatment with Hanks' balanced salt solution or NaCl lowered excystation; however, this effect was overcome with acid. Sodium ions were replaceable with potassium ions, and sodium bicarbonate was replaceable with sodium phosphate. Oocysts that received 200 krad irradiation excysted at the same rates as nonirradiated oocysts (95%), the excystation rates were lowered (50%) by 2,000 krad, and no excystation was observed by 5,000 krad. No differences were observed between the propidium iodide (PI) permeability of untreated oocysts and oocysts treated with 200 krad, while 92% of oocysts were PI positive after 2,000 krad. Most of the sporozoites exposed to 2,000 krad were not viable as indicated by the dye permeability assay. The oocysts irradiated with 200 and 2,000 krad infected cells, but no replication was observed. The results suggest that gamma-irradiated oocysts may still be capable of excystation and apparent infection; however, because the sporozoites could not reproduce they must not have been viable.     

Co-localization of epithelial sodium channels and glutamate receptors in single taste cells

Umami taste is elicited by monosodium glutamate (MSG), a compound consisting of two potent taste stimuli, Na(+) and glutamate. In rat fungiform taste cells, amiloride-sensitive epithelial sodium channels (ENaCs) mediate Na(+) transduction, while glutamate is transduced by a combination of ionotropic and metabotropic glutamate receptors. We used giga-seal whole-cell recording to determine if responses to glutamate and Na(+) occur in the same taste cells. Approximately 68% of the cells tested responded to amiloride, indicating that they express functional ENaCs. Responses to glutamate occurred in about 58% of the cells tested. Interestingly, responses to glutamate occurred in the subset of cells that also responded to amiloride, indicating that glutamate receptors are located preferentially in the same taste cells that also express ENaCs. Further experiments showed that amiloride did not suppress responses to glutamate under voltage-clamp conditions. Taken together, the data suggest that although ENaCs are not involved directly in glutamate transduction, their co-localization with glutamate receptors provides a substrate for the cellular integration of these independent pathways. Copyright Copyright 1999 S. Karger AG, Basel     

Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

The DNA binding stringency of restriction endonucleases is crucial for their proper function. The X-ray structures of the specific and non-cognate complexes of the restriction nuclease EcoRV are considerably different suggesting significant differences in the hydration and binding free energies. Nonetheless, the majority of studies performed at pH 7.5, optimal for enzymatic activity, have found a < 10-fold difference between EcoRV binding constants to the specific and nonspecific sequences in the absence of divalent ions. We used a recently developed self-cleavage assay to measure EcoRV-DNA competitive binding and to evaluate the influence of water activity, pH and salt concentration on the binding stringency of the enzyme in the absence of divalent ions. We find the enzyme can readily distinguish specific and nonspecific sequences. The relative specific-nonspecific binding constant increases strongly with increasing neutral solute concentration and with decreasing pH. The difference in number of associated waters between specific and nonspecific DNA-EcoRV complexes is consistent with the differences in the crystal structures. Despite the large pH dependence of the sequence specificity, the osmotic pressure dependence indicates little change in structure with pH. The large osmotic pressure dependence means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowded environment of the cell. In addition to divalent ions, water activity and pH are key parameters that strongly modulate binding specificity of EcoRV.     

Importance of extracellular divalent cations to polarisation of polymorphonuclear leukocytes induced by plasma

The roles of extracellular calcium and magnesium ions in the polarisation of human peripheral blood polymorphonuclear leukocytes (PMN) induced by autologous fresh heparinised plasma were investigated by studying the effects of 5 mM chelators of divalent cations [ethylenediamine tetra-acetic acid (EDTA), ethylenebis-(oxyethylene-nitrilo)-tetra-acetic acid (EGTA) or disodium hydrogen citrate]. In addition, the effects of a blocker of membrane calcium channels (verapamil) were studied. Polarisation of PMN suspended in plasma (84.1 +/- 11.9%) was reduced by each chelating agent over 30 min (to 20.0 +/- 15.6% by EDTA, to 42.5 +/- 19.3% by EGTA and to 29.4 +/- 22.9% by citrate). Polarisation of PMN suspended in plasma treated with EDTA or EGTA was restored by inclusion of equimolar additional Ca2+ ions, and in plasma treated with EDTA, EGTA or citrate, by equimolar additional Mg2+ ions. Additional Mg2+ had no effect on the spherical shape of PMN in Hanks' solution and additional cations had no effects on the polarisation of PMN induced by fMLP. Cells rendered spherical by each chelating agent in plasma for 30 min retained their ability to polarise on addition of fMLP to the plasma-chelator medium. Verapamil (10(-4) M) markedly reduced polarisation in plasma (to 52 +/- 11.3%) but the same drug (10(-5) M) had no such effect. In contrast to the polarisation of cells in plasma, the polarisation response of PMN to N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10(-8) M) in buffered Hanks' solution was not affected by any of the chelating agents or by verapamil, even in high concentration. These results indicate that extracellular divalent cations are necessary for the polarisation of PMN suspended in autologous plasma and that the mechanism of polarisation of PMN in plasma may be different to that of polarisation induced by fMLP.     

Evidence that alterations in small molecule permeability are involved in the Clostridium perfringens type A enterotoxin-induced inhibition of macromolecular synthesis in Vero cells

The mechanism by which Clostridium perfringens enterotoxin (CPE) simultaneously inhibits RNA, DNA, and protein synthesis is unknown. In the current study the possible involvement of small molecule permeability alterations in CPE-induced inhibition of macromolecular synthesis was examined. Vero cells CPE-treated in minimal essential medium (MEM) completely ceased net precursor incorporation into RNA and protein within 15 minutes of CPE treatment. However, RNA and protein synthesis continued for at least 30 minutes in Vero cells CPE-treated in buffer (ICIB) approximating intracellular concentrations of most ions. Addition of intracellular concentrations of amino acids to ICIB (ICIB-AA) caused a further small but detectable increase in protein synthesis in CPE-treated cells. ICIB did not affect CPE-specific binding levels or rates. Similar small molecule permeability changes (i.e., 86Rb-release) were observed in cells CPE-treated in either ICIB or in Hanks' balanced salt solution. Collectively these findings suggest that CPE-treatment of cells in ICIB-AA ameliorates CPE-induced changes in intracellular concentrations of ions and amino acids and permits the continuation of RNA and protein synthesis. These results are consistent with and support the hypothesis that permeability alterations for small molecules are involved in the CPE-induced inhibition of precursor incorporation into macromolecules in Vero cells.     

Biochemical studies of taste sensation. IX. Enhancement of L-[3H]glutamate binding to bovine taste papillae by 5'-ribonucleotides

The interaction of the taste substance monosodium L-glutamate with taste receptors has been investigated. Binding of L-[3H]glutamate was measured to preparations of bovine circumvallate (taste) papillae (type I preparation) and to control tongue epithelial preparations (type II preparation) devoid of taste receptors. Binding is operationally defined using a membrane filtration assay. Substantially greater binding occurred to the type I preparation than to the type II preparation, and the binding to the type I preparation showed evidence of saturation. The apparent Kd of L-glutamate was estimated to be in the range of 20--30 mM. The unique taste effect of L-glutamate was considered to depend importantly on its demonstrated synergism in combination with certain 5'-ribonucleotides. A several-fold enhancement of binding of L-[3H]glutamate occurred in the presence of certain 5'-ribonucleotides. 5'-GMP, 5'-IMP and 5'-UMP each increased the binding of L-[3H]glutamate, while 5'-XMP, 5'-AMP and 5'-CMP did not. None of these nucleotides affected the lower level of binding to the type II preparation. Neither the free bases, adenine and guanine, their nucleosides nor their di- or triphosphonucleotides were effective in increasing L-[3H]glutamate binding to the type I preparation. The nucleotide specificity of the glutamate binding enhancement therefore shows a marked similarity with the nucleotide specificity in evoking the synergistic taste effect in humans.     

Polymorphonuclear leukocyte locomotion is insensitive to lowered cytoplasmic calcium levels

Chemotactic factors stimulate the rate of locomotion of polymorphonuclear leukocytes (PMNs). To investigate the importance of cytoplasmic calcium we have examined the ability of the chemotactic peptide N-formylnorleucyl eucylphenalanine (FNLLP) to stimulate the locomotion of PMNs whose cytoplasmic calcium levels were reduced by incubation in EGTA or in EGTA plus the calcium ionophores, ionomycin or A23187. Locomotion was assayed by migration through micropore filters and by time-lapse videomicroscopy. Cells in EGTA exhibited similar or slightly reduced rates of locomotion compared to cells in Hanks' balanced salt solution (HBSS). The peptide dose dependence for the stimulation of locomotion was similar in medium containing calcium or EGTA. The presence of 1 microM ionophore plus EGTA had no effect on the stimulation of locomotion by peptide. The presence of ionophores (1 microM) plus external calcium inhibited locomotion.     

Rapid formation of tight junctions in HT 29 human adenocarcinoma cells by hypertonic salt solutions

The human colon adenocarcinoma cell line HT 29 grows virtually without tight junctions (TJ) under standard culture conditions. Earlier studies have shown that focal TJ (fasciae occludentes) can be rapidly assembled in this cell line under the influence of various proteases. Here we show that focal TJ can be induced in this cell line by a brief treatment with appropriate salt solutions. Induction by ammonium sulfate in Hanks' buffer reached a maximum value after 15 to 30 min. The amount of TJ increased with the salt concentration and reached a plateau value at a concentration of 160 mM ammonium sulfate. The amount and complexity of TJ induced by ammonium sulfate were similar to those in experiments using trypsin as inducing agent as shown by morphometric analysis. At 0 degrees C, no TJ were formed under the influence of the salt. A comparative study of TJ induction using a variety of inorganic and organic salts gave the following results. All alkali sulfates induced TJ, although with different yield. Both calcium and magnesium chloride were potent inducers. Ammonium and sodium salts encompassing a variety of anions covered a wide range from maximum induction (sulfate, citrate) to almost complete absence of induction (nitrate). Sodium chloride did not induce any TJ. It follows that the induction of TJ is a specific effect of individual ionic components of the solution as opposed to a general effect of osmolarity and ionic strength. The data suggest tentatively that antichaotropic but not chaotropic ions have the potential to trigger the formation of TJ in this experimental system.     

Changes in the morphology of Ehrlich ascites tumour cells caused by hyperosmotic media

It was found that Ehrlich ascites tumour cells undergo significant morphology changes in media in which osmolarity is increased by NaCl or Hanks' balanced salt solution. The morphology changes include formation of filopodia- and lamellipodia-like cell surface protrusions. Their formation is enhanced by an addition of cytochalasin B. The data obtained suggest that both changes in plasma membrane properties and changes in activity of contractile apparatus participate in the formation of cell surface protrusions.     

Stabilization of soluble active rat liver glucagon receptor

Active glucagon receptor was solubilized with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (Chaps) from rat liver plasma membranes but rapidly (less than 8 h) lost activity. Either inclusion of 1X Hanks' balanced salt solution in the 3 mM Chaps solubilization buffer or its addition after solubilization increased the percentage of total binding attributable to specific glucagon binding from approximately 10 to greater than 80%; of great importance, it increased the stability from near zero binding at 8 h to 50% binding at 48 h (4 degrees C). Of the Hanks' solution components, either NaCl (137 mM) or CaCl2 (1.26 mM) was effective in increasing specific binding to approximately 70 and 60% respectively: Mg salts were ineffective. Soluble receptor binding activity was assayed by dextran-coated charcoal adsorption of free hormone. The assay is rapid, simple, and reproducible. It is suitable for monitoring receptor activity during purification and molecular characterization. Competition binding studies gave an IC50 value of 10-20 nM (slope factor approximately 1), with or without GTP. Dissociation assays revealed GTP sensitivity when receptors were solubilized either as glucagon-receptor complexes or free receptor. Active glucagon-receptor complexes could be eluted from wheat germ lectin-agarose: neither concanavalin A-agarose nor soybean agglutinin-agarose bind receptor. A glucagon degrading activity which co-solubilized with the receptor but did not require detergent for extraction was distinguishable from the soluble receptor not only by solubility but also by its heat stability (30 degrees C), its inhibition by bacitracin, its affinity for glucagon, its retention of activity for at least 1 week at 4 degrees C, and its size.     

Adhesion of rat hepatocytes to collagen

Rat hepatocytes, freshly isolated with a collagenase perfusion technique, were found to attach within 1 h on collagen substrates and on culture dishes coated with cold insoluble globulin (CIG) or asialoceruloplasmin (AC). Spreading was observed on collagen and CIG but not on AC. Both attachment and spreading occurred in a simple balanced salt solution in the absence of serum. In the absence of serum no attachment was observed on plain plastic dishes or on dishes coated with serum albumin or other plasma proteins, unless divalent manganese ions were present. In the presence of manganese the hepatocytes attached to all surfaces tested, but no spreading occurred. Attachment to collagen occurred equally well to collagens type I or type III both in the native, fibrillar state and in the denatured state. Collagen attachment required magnesium ions but did not appear to involve the collagen-linked carbohydrates. Different mechanisms were found to operate in hepatocyte attachment to collagen and to AC; the latter is most likely mediated by the hepatocyte surface receptor involved in recognition and uptake of asialoglycoproteins. The role of CIG in hepatocyte attachment to collagen was investigated. Data are presented suggesting that this glycoprotein, which mediates the adhesion of fibroblasts to collagen, is not required for hepatocyte attachment to collagen.     

Glutamate may be an efferent transmitter that elicits inhibition in mouse taste buds

Recent studies suggest that l-glutamate may be an efferent transmitter released from axons innervating taste buds. In this report, we determined the types of ionotropic synaptic glutamate receptors present on taste cells and that underlie this postulated efferent transmission. We also studied what effect glutamate exerts on taste bud function. We isolated mouse taste buds and taste cells, conducted functional imaging using Fura 2, and used cellular biosensors to monitor taste-evoked transmitter release. The findings show that a large fraction of Presynaptic (Type III) taste bud cells (～50%) respond to 100 μM glutamate, NMDA, or kainic acid (KA) with an increase in intracellular Ca(2+). In contrast, Receptor (Type II) taste cells rarely (4%) responded to 100 μM glutamate. At this concentration and with these compounds, these agonists activate glutamatergic synaptic receptors, not glutamate taste (umami) receptors. Moreover, applying glutamate, NMDA, or KA caused taste buds to secrete 5-HT, a Presynaptic taste cell transmitter, but not ATP, a Receptor cell transmitter. Indeed, glutamate-evoked 5-HT release inhibited taste-evoked ATP secretion. The findings are consistent with a role for glutamate in taste buds as an inhibitory efferent transmitter that acts via ionotropic synaptic glutamate receptors.