ATP has been shown to be a taste bud afferent transmitter, but the cells responsible for, and the mechanism of, its release have not been identified. Using CHO cells expressing high-affinity neurotransmitter receptors as biosensors, we show that gustatory stimuli cause receptor cells to secrete ATP through pannexin 1 hemichannels in mouse taste buds. ATP further stimulates other taste cells to release a second transmitter, serotonin. These results provide a mechanism to link intracellular Ca 2؉ release during taste transduction to secretion of afferent transmitter, ATP, from receptor cells. They also indicate a route for cell-cell communication and signal processing within the taste bud.afferent ͉ gustation ͉ serotonin ͉ synapses G ustatory receptor cells within taste buds detect sweet, bitter, and umami tastants via G protein-coupled taste receptors. Although detailed transduction mechanisms downstream of such receptors have been elucidated (1), our understanding of the signaling from taste cells to the afferent nerve is still limited. ATP has emerged as a key afferent neurotransmitter for taste buds (2). Gustatory stimulation of taste buds also results in release of serotonin (5-HT) (3). Yet, which cells release each neurotransmitter and the mechanisms of such release are unknown. These problems are particularly enigmatic, because in taste buds, the cells that express taste receptors (i.e., ''receptor cells'') comprise a separate population from the cells that possess synapses, express synaptic proteins, and exhibit depolarizationdependent calcium influx (''presynaptic cells'') (4-6). We have used cellular biosensors (3) to measure taste-evoked transmitter release and, particularly, to identify which cells secrete ATP and 5-HT. Our results show that only receptor cells release ATP and only presynaptic cells release 5-HT. Further, we demonstrate an unexpected mechanism for nonexocytotic ATP secretion and present evidence for cell-cell signaling between receptor and presynaptic cells upon taste stimulation. ResultsWe isolated taste cells from mouse circumvallate papillae, loaded them with the Ca 2ϩ indicator Fura2-AM, and measured responses to taste stimulation and to KCl depolarization. Concurrently, we also measured transmitter release from individual taste cells using cellular biosensors (see below). Taste cells were unambiguously identified either as receptor cells or presynaptic cells by whether they responded to taste stimulation (receptor cells) or to KCl depolarization (presynaptic cells) (4). Isolated receptor and presynaptic cells were present in sufficiently low density in the recording chamber that there were no interactions (e.g., diffusible signals) between individual taste cells. The only interactions measured were between an isolated taste cell and its apposed biosensor. Taste Receptor Cells Secrete ATP via Gap Junction Hemichannels.When a Fura2-loaded CHO cell stably expressing P2ϫ2/ P2ϫ3 receptors (hereafter, ''ATP biosensor'') was positioned in close proximity to a receptor cell (Fig. 1A), we ...

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By human sensory analyses, we found that various extracellular calcium-sensing receptor (CaSR) agonists enhance sweet, salty, and umami tastes, although they have no taste themselves. These characteristics are known as "kokumi taste" and often appear in traditional Japanese cuisine. Although GSH is a typical kokumi taste substance (taste enhancer), its mode of action is poorly understood. Here, we demonstrate how the kokumi taste is enhanced by the CaSR, a close relative of the class C G-protein-coupled receptors T1R1, T1R2, and T1R3 (sweet and umami receptors). We identified a large number of CaSR agonist ␥-glutamyl peptides, including GSH (␥-Glu-Cys-Gly) and ␥-Glu-Val-Gly, and showed that these peptides elicit the kokumi taste. Further analyses revealed that some known CaSR agonists such as Ca 2؉ , protamine, polylysine, L-histidine, and cinacalcet (a calcium-mimetic drug) also elicit the kokumi taste and that the CaSR-specific antagonist, NPS-2143, significantly suppresses the kokumi taste. This is the first report indicating a distinct function of the CaSR in human taste perception.

A dissipative particle dynamics ͑DPD͒ simulation has been used to study the spontaneous vesicle formation of amphiphilic molecules in aqueous solution. The amphiphilic molecule is represented by a coarse-grained model, which contains a hydrophilic head group and a hydrophobic tail. Water is also modeled by the same size particle as adopted in the amphiphile model, corresponding to a group of several H 2 O molecules. In the DPD simulation, from both a randomly dispersed system and a bilayer structure of the amphiphile for the initial condition, a spontaneous vesicle formation is observed through the intermediate state of an oblate micelle or a bilayer membrane. The membrane fluctuates and encapsulates water particles and then closes to form a vesicle. During the process of vesicle formation, the hydrophobic interaction energy between the amphiphile and water is diminishing. It is also recognized that the aggregation process is faster in two-tailed amphiphiles than those in the case of single-tailed ones.

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Synapses between gustatory receptor cells and primary sensory afferent fibers transmit the output signal from taste buds to the CNS. Several transmitter candidates have been proposed for these synapses, including serotonin (5-HT), glutamate, acetylcholine, ATP, peptides, and others, but, to date, none has been unambiguously identified. We used Chinese hamster ovary cells stably expressing 5-HT 2C receptors as biodetectors to monitor 5-HT release from taste buds. When taste buds were depolarized with KCl or stimulated with bitter, sweet, or sour (acid) tastants, serotonin was released. KCl-and acid-induced 5-HT release, but not release attributable to sweet or bitter stimulation, required Ca 2ϩ influx. In contrast, 5-HT release evoked by sweet and bitter stimulation seemed to be triggered by intracellular Ca 2ϩ release. These experiments strongly implicate serotonin as a taste bud neurotransmitter and reveal unexpected transmitter release mechanisms.

Taste buds contain two types of cells that directly participate in taste transduction -receptor (Type II) cells and presynaptic (Type III) cells. Receptor cells respond to sweet, bitter and umami taste stimulation but until recently the identity of cells that respond directly to sour (acid) tastants has only been inferred from recordings in situ, from behavioural studies, and from immunostaining for putative sour transduction molecules. Using calcium imaging on single isolated taste cells and with biosensor cells to identify neurotransmitter release, we show that presynaptic (Type III) cells specifically respond to acid taste stimulation and release serotonin. By recording responses in cells isolated from taste buds and in taste cells in lingual slices to acetic acid titrated to different acid levels (pH), we also show that the active stimulus for acid taste is the membrane-permeant, uncharged acetic acid moiety (CH 3 COOH), not free protons (H + ). That observation is consistent with the proximate stimulus for acid taste being intracellular acidification, not extracellular protons per se. These findings may also have implications for other sensory receptors that respond to acids, such as nociceptors.

The structure of C6o molecules adsorbed on the Cu(l 1 l)-(l x 1) surface has been investigated by scanning tunneling microscopy (STM). Bias voltage dependent STM images of individual C6o molecules in the monolayer film showed unique intramolecular structures with a threefold symmetry. The observed images agree well with those calculated using the local density approximation. With charge transfer from the Cu(l 11) substrate to the monolayer film, the €50 molecule ratchets to threefold hollow sites. PACS numbers: 68.35.Bs, 61.16.Ch, 61.46,+w, 68.65.+g The investigation on carbon fullerenes discovered by Kroto et al. [1] has been a growing exciting field, since Kratschmer et al. [2] succeeded in the extraction and purification of C60 and other fullerenes. Discovery of superconductivity by Hebard et al. [3] in a K-doped fullerene sample, K3C60, added further excitement with promising practical applications [4]. Scanning tunneling microscopy (STM) has been successfully used to reveal the geometric and electronic structure of the fullerenes with molecular resolution. The nucleation and growth of C60 film and its structure on various substrates, such as Au(100) [5], (111) and (110) [6], Ag(lll) [7], highly oriented pyrolytic graphite (0001) [8], GaAs(llO) [9], and Si(l 11) [10], and (100) [11] have been major interests in STM studies. Investigations on other fullerene films, such as C70 [12], Cu [13], and SCC74/SC2C74 [14], and Sc 2 C 8 4 [15] on the Si (100)-(2x 1) surface have been reported using the FI-STM (field ion scanning tunneling microscope), elucidating the growth kinetics and geometric structures.The Cu(l 11) surface is one of the ideal surfaces for the growth of C60 film similar to the bulk [16], since the lattice mismatch is small (2%) between the nearest neighbor (nn) distance (10.0 A) of the bulk Ceo crystal and 4 times of the Cu-Cu nn distance (10.2 A). A recent study of C 60 adsorption on the Cu(lll), (110), and (100) surfaces using x-ray photoemission spectroscopy (XPS) and high resolution electron energy loss spectroscopy (HREELS) and other techniques [17] concluded the following: (1) The Cu substrate donates charge to the lowest unoccupied molecular orbital (LUMO) band of C60 as K does in K x C6o films and (2) single-domain epitaxy with a (4x4) superlattice and successive layer-bylayer growth can be achieved on the Cu(l 11) surface.In this Letter, we report the intramolecular structure of C60 molecules on the Cudll)-(lxl) surface using the FI-STM [18]. C60 molecules ratchet to hollow sites and are ordered due to the substrate-C6o and C60-C60 interactions. Observed intramolecular structure with a unique threefold symmetry agrees well with the charge density around the molecule calculated using the local density approximation (LDA). It was confirmed in the STM images and calculated charge density that excess charge on the molecule is present on top of the pentagonal rings.The experimental details of the extraction and purification of high-purity C60 powder (purity of 99.95%, the rest being C70) [19], a...