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We previously showed that the hypertonicity of taste stimulating solutions modified tonic responses, the quasi-steady state component following the transient (phasic) component of each integrated taste nerve response. Here we show that the hypertonicity opens tight junctions surrounding taste receptor cells in a time-dependent manner and modifies whole taste nerve responses in bullfrogs. We increased the tonicity of stimulating solutions with non-taste substances such as urea or ethylene glycol. The hypertonicity enhanced phasic responses to NaCl>0.2M, and suppressed those to NaCl<0.1M, 1mM CaCl2, and 1mM bitter substances (quinine, denatonium and strychnine). The hypertonicity also enhanced the phasic responses to a variety of 0.5M salts such as LiCl and KCl. The enhancing effect was increased by increasing the difference between the ionic mobilities of the cations and anions in the salt. A preincubation time >20s in the presence of 1M non-taste substances was needed to elicit both the enhancing and suppressing effects. Lucifer Yellow CH, a paracellular marker dye, diffused into bullfrog taste receptor organs in 30s in the presence of hypertonicity. These results agreed with our proposed mechanism of hypertonic effects that considered the diffusion potential across open tight junctions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We show here the expression, permeability and pharmacology of a voltage-gated channel in certain taste bud cells (TBCs) which is known to be permeable to Lucifer Yellow CH (LY) and known to release ATP as a neurotransmitter in response to taste substances. LY dissolved in a 200 mM K(+)-containing solution label TBCs immunoreactive to PLCβ2, a phospholipase subtype, but not the TBC subtype immunoreactive to SNAP-25, a SNARE protein. In addition to these subtypes, LY also labelled a few of the non-immunoreactive TBCs. Monovalent and divalent anion probes with molar mass less than 1200 also label PLCβ2-immunoreactive TBCs and a few non-immunoreactive TBCs, whereas a cation probe, rhodamine B, labels the cell membrane of TBCs nonselectively and K(+) independently. The number of LY-labelled TBCs is decreased by 5 μM DIDS (4,4'-diisothiocyanostilbene-2-2'disulfonate), 1mM octanol and 10(-5)M H(+), but not by 10 μM carbenoxolone, 2mM probenecid, 10mM TEA, or 30 μM flufenamic acid. PLCβ2-immunoreactive TBCs and a few non-immunoreactive TBCs generate a TEA-insensitive outwardly rectifying current. DIDS decreases this current in magnitude with IC(50) of ~0.4 μM in a voltage-independent manner. Also 10(-5)M H(+) and 1mM octanol decreases the current magnitude, but 10 μM carbenoxolone and 2mM probenecid do not. These results show that the LY-permeable channel preferably permeates anions and occurs not only on PLCβ2-immunoreactive TBCs but also on certain non-immunoreactive TBCs. Also the results show that the pharmacology of the LY-permeable channel is different from hemichannels reported. The discussion focuses on the pharmacology and the role of the LY-permeable channel., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Mammalian taste bud cells (TBCs) consist of several cell types equipped with different taste receptor molecules, and hence the ratio of cell types in a taste bud constitutes the taste responses of the taste bud. Here we show that the population of immunohistochemically identified cell types per taste bud is proportional to the number of total TBCs in the taste bud or the area of the taste bud in fungiform papillae, and that the proportions differ among cell types. This result is applicable to soft palate taste buds. However, the density of almost all cell types, the population of cell types divided by the area of the respective taste buds, is significantly higher in soft palates. These results suggest that the turnover of TBCs is regulated to keep the ratio of each cell type constant, and that taste responsiveness is different between fungiform and soft palate taste buds., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Lucifer Yellow CH (LY), a fluorescent membrane-impermeable cell-marker dye, has been routinely loaded into cells through recording electrodes to visualize these cells after electrophysiological investigation. Recently we showed that LY produced superoxide when LY was exposed to light at ordinary intensities for microscopy, and that the resultant superoxide retarded the inactivation of voltage-gated Na+ channels even in the dark. Here, we show that superoxide produced by exposure to light prolongs the duration of action potentials, and increases the magnitude of outward rectifier K+ currents and inward rectifier K+ currents in cultured mouse hippocampal neurons. Superoxide also increases the current response of AMPA receptors, but has no effect on that of kainate and NMDA receptors, GABA(A) receptors, high-voltage activated Ca2+ channels of the hippocampal neurons, nor on 5HT3 receptors of N1E-115 cells. These superoxide effects are irreversible. The addition of superoxide dismutase, an enzyme that selectively decomposes superoxide, to LY-loaded recording electrodes reverses the superoxide effects, but addition of heat-inactivated superoxide dismutase fails to reverse the effects. The application of dithiothreitol, a free radical scavenger, to a bathing solution also reverses the superoxide effects. This shows that superoxide rather selectively modifies ion channels. The effects of exogenous and endogenous superoxide on the superoxide-susceptible channels are discussed.

Taste bud cells (TBCs) on soft palates differ from those on tongues in innervation and chemosensitivity. We investigated voltage-gated channels involved in the taste responses of TBCs on mouse soft palates under in-situ tight-seal voltage/current-clamp conditions. Under the cell-attached mode, TBCs spontaneously fired action currents, which were blocked by application of 1 microM TTX to TBC basolateral membranes. Firing frequencies increased in response to taste substances applied to TBC receptor membranes. Under the whole-cell clamp mode, as expected, TBCs produced various voltage-gated currents such as TTX-sensitive Na+ currents (INa), outward currents (Iout) including TEA-sensitive and insensitive currents, inward rectifier K+ currents (Iir), and Ca2+ currents including T-type, P/Q-type, and L-type Ca2+ currents. We classified TBCs into three types based on the magnitude of their voltage-gated Na+ currents and membrane capacitance. HEX type (60% of TBCs examined) was significantly larger in Na+ current magnitude and smaller in membrane capacitance than LEX type (23%). NEX type (17%) had no Na+ currents. HEX type was equally distributed within single taste buds, while LEX type was centrally distributed, and NEX type was peripherally distributed. There were correlations between these electrophysiological cell types and morphological cell types determined by three-dimensional reconstruction. The present results show that soft palate taste buds contain TBCs with different electrophysiological properties, and suggest that their co-operation is required in taste transduction.

The carp gustatory responses to various salts and those to distilled water after adaptation of the receptors to the salts were recorded from the palatine nerve under varying conditions. (a) As far as Na4Fe(CN)6 solution prepared freshly was used, any peak in the dose-response curve was not observed, while the solution stored overnight induced a large peak response at the low concentration region as Konishi reported. The magnitude of the water response after adaptation to the stored solution was practically equal to that after adaptation to the fresh solution, suggesting that the receptor site for the water response is different from that for the dilute salts. (b) The responses to salts depended largely on the species of both cations and anions of the salts. The responses were deceased with an increase in the lyotropic number of the anions and increased with an increase in the number above 11. The response to distilled water was practically independent of the species of both monovalent cations and anions of the salts used for the adaptation. The pH dependence of the response to 10 mM NaCl was largely different from that to distilled water after adaptation to 10 mM NaCl. (c) The water response was suppressed by the presence of electrolytes in stimulating solution; the data obtained with different species of salts were described by a single curve as a function of the ionic strength. (d) The mechanism to explain how distilled water leads to depolarization of the taste cell was discussed in terms of the electric double-layer potential.

The effects of changed ionic environments on the taste responses to amino acids were examined by recording the activity of the palatine nerve. The responses to glycine, L-proline and L-arginine dissolved in deionized water were greatly reduced after ions were removed from the surface of the palatine epithelium by treatment with 5 mM EDTA. The addition of various species of salts to amino acid solutions caused reversible recovery of the responses. The response to 0.1 mM glycine increased with an increase in salt concentration in amino acid solutions and reached saturation level. The effects of salts of divalent cations (CaCl2 and MgCl2) appeared at much lower concentration (10(-7) M) than those of salts of monovalent cations (NaCl and KCl) (10(-4) M), suggesting that cations support the taste response. All the organic cations examined, including those of large molecular weights (choline, Tris, bis-Tris, bis-Tris propane, tetraethylammonium, triethanolamine and D-glucosamine), also supported the taste responses. The results obtained led to a conclusion that not a specific cation but various species of cations can support the eel taste responses. It is suggested that the cations do not act as current carrying ions to depolarize the taste cells but that the binding of the cations to the receptor membrane plays an essential role in taste reception.

The effects of changed ionic environments on the olfactory responses in the carp, the rainbow trout, and the bullfrog were examined by recording the stimulant-induced waves from the olfactory bulb. (a) Application of stimulants (various species of amino acids and beta-ionone) dissolved in deionized water to the EDTA-treated olfactory epithelium of the carp did not induce any response. The addition of various species of salts to the stimulating solution reversibly restored the response. (b) The responses of the carp to L-alanine in the presence of MgCl2 and MgSO4 and those in the presence of KCl and K4Fe(CN)6 are described by single curves, respectively, as a function of concentration of the cations, suggesting that the cations support olfactory reception. (c) All the inorganic cations examined (Li+, NH3+, K+, Ca2+, Mg2+, Co2+, Mn2+, Cd2+) and organic cations (Tris+, choline+, bis-Tris propane2+) were effective to support the response of the carp, whereas TEA+, TBA+, triethanolamine+, and bis-Tris+ were ineffective. (d) The olfactory responses of the rainbow trout and the bullfrog were also reduced by removal of ions from the surface of the epithelia and recovered by addition of ions. (e) It is suggested that the cations do not act as current carriers across the apical olfactory cell membrane for the generation of the receptor potential.

A quantitative method of cross-adaptation was developed to explore the difference in the receptor sites for various amino acids in the carp olfactory receptors. The olfactory responses were measured by recording the stimulant-induced waves from the olfactory bulb. Cross-adaptation was carried out by varying concentrations of amino acids in a wide range. Typical examples of the results obtained are as follows. The response to Thr after Ser was decreased with increasing concentration of Ser applied first and reached the spontaneous level, while that to Thr after Glu was decreased to 77% of the level of the original response with increasing Glu concentration and stayed this constant level with a further increase in Glu concentration. Application of the amino acids in the reverse order gave essentially similar results. Such types of experiments were carried out between various pairs of amino acids. The results obtained suggested that amino acids of very close species (e.g. Thr and Ser, Asp and Glu, Tyr and Phe) stimulate the same respective receptor sites and that amino acids of most other pairs stimulate more or less different receptor sites, although there exist the receptor sites stimulated commonly by both amino acids of one pair. It was concluded that the carp olfactory cells have many different receptor sites stimulated only by one species of amino acid and its close analogues.

Synergism between 5'-nucleotides and L-amino acids in the rat taste nerve responses was investigated. The synergism was examined between low concentration of the nucleotides which induce only a negligibly small response and various concentrations of amino acids. A marked synergism was found between 5'-nucleotides and all the amino acids examined. Purine 5'-nucleotides such as GMP, deoxy GMP, GDP, GTP, AMP or IMP exhibited the synergistic effect on the responses to amino acids, while pyrimidine 5'-nucleotides such as CMP or UMP exhibited practically no synergistic effect. The presence of GMP led to a shift of the concentration-response curves for amino acids to a lower concentration region without affecting the response of the saturation level. These curves were analyzed under the assumption that there exist two types of receptor sites with different dissociation constants for each amino acid. The results suggest that GMP leads to a decrease in dissociation constants, especially for high affinity sites without affecting maximal responses. Monosodium glutamate (Glu) itself, which is known as a typical 'flavor potentiator', exhibited no enhancing effect on the responses to 4 primary taste stimuli. The mechanism of how Glu and the 5'-nucleotides 'potentiate' flavor of food is discussed.

In this study, in vitro synthesized mRNA encoding mouse and Torpedo nicotinic acetylcholine receptor subunits was injected into Xenopus oocytes, followed by assays for assembly onto the oocyte surface (using [125I]alpha-bungarotoxin binding) and for acetylcholine-induced conductances (using voltage clamp). We constructed hybrid acetylcholine receptors in Xenopus oocytes by injecting all 8 possible combinations of 4 subunit-specific mRNAs in which a single subunit is derived from the other species. For each hybrid combination, there is detectable assembly and conductance. We also constructed cDNA clones that encode chimeric acetylcholine receptor subunits in which part of the gamma subunit from Torpedo was replaced by the homologous region of the delta subunit from mouse. None of the chimeric subunits was able to replace the Torpedo gamma, mouse delta, or Torpedo delta subunit with regard to assembly or function. We therefore conclude that widely spaced (and unknown) parts of the protein chain are required for the intersubunit interactions that eventually lead to functional assembly of the receptor.