Patch clamp recording of the responses to three bitter stimuli in mouse taste cells.

Although several pathways of bitter taste signal transduction have been proposed in taste cells, these mechanisms have not been elucidated in detail. To investigate the diversity of responses to bitter stimuli, we recorded the electrophysiological responses to quinine, denatonium and naringin using whole-cell patch clamp technique in isolated taste cells of C57BL/6J mice. Ten mM quinine induced depolarizing response under the current clamp mode, and inward current response under the voltage-clamp mode (holding potential -80 mV) using both K+ (with pseudo intracellular solution) and Cs+ (K+ was substituted by Cs+ in the pseudo intracellular solution) pipettes. However, when the K+ pipette was used, the membrane conductance was suppressed and activated in succession. On the other hand, the membrane conductance was only activated when the Cs+ pipette was used. Half to one mM denatonium induced depolarizing response under the current clamp mode, and outward current response under the voltage clamp mode with both pipettes. Using these pipettes, the membrane conductance was activated or suppressed in the individual case. Naringin-induced responses were not detected in these measurements. These electrophysiological recordings suggest that multiple transduction mechanisms are involved in bitter taste perception in mouse taste cells.     

Differential covariation in taste responsiveness to bitter stimuli in rats

Variation exists in the sensitivity of individual rodents and humans to different bitter tastants. An absence of uniform correlation in responsiveness to different bitter substances across individuals within a species suggests heterogeneity in the mechanisms underlying stimulus processing within this taste modality. Here, we examined taste responsiveness of individual rats to three bitter compounds (quinine hydrochloride, denatonium benzoate, and cycloheximide) in short-term lick tests to determine the magnitude of covariation among responses to these stimuli and infer commonalities in their receptor and neural mechanisms. Rats were tested with a given pair of bitter stimuli during three sessions comprising randomized trial blocks of six concentrations of each stimulus + deionized water. Psychophysical functions were generated for individual rats for respective stimulus pairs, and concentrations of each stimulus that produced equivalent lick suppression relative to water were correlated across animals. Behavioral taste responsiveness to quinine hydrochloride strongly covaried with responsiveness to denatonium benzoate (r = +0.82). Lick responsiveness to quinine was less robustly correlated with that to cycloheximide (r = +0.44), and denatonium and cycloheximide responses failed to correlate. These results imply substantial overlap in the bitter taste coding mechanisms for quinine and denatonium but some degree of independence in the mechanisms responsible for gustatory processing of cycloheximide. More generally, these data reinforce the notion that bitter taste processing is not a homogeneous event.     

Tastants evoke cAMP signal in taste buds that is independent of calcium signaling

We previously showed that rat taste buds express several adenylyl cyclases (ACs) of which only AC8 is known to be stimulated by Ca²⁺. Here we demonstrate by direct measurements of cAMP levels that AC activity in taste buds is stimulated by treatments that elevate intracellular Ca²⁺. Specifically, 5 µM thapsigargin or 3 µM A-23187 (calcium ionophore), both of which increase intracellular Ca²⁺ concentration ([Ca²⁺]_i), lead to a significant elevation of cAMP levels. This calcium stimulation of AC activity requires extracellular Ca²⁺, suggesting that it is dependent on Ca²⁺ entry rather than release from stores. With immunofluorescence microscopy, we show that the calcium-stimulated AC8 is principally expressed in taste cells that also express phospholipase C₂ (i.e., cells that elevate [Ca²⁺]_i in response to sweet, bitter, or umami stimuli). Taste transduction for sucrose is known to result in an elevation of both cAMP and calcium in taste buds. Thus we tested whether the cAMP increase in response to sucrose is a downstream consequence of calcium elevation. Even under conditions of depletion of stored and extracellular calcium, the cAMP response to sucrose stimulation persists in taste cells. The cAMP signal in response to monosodium glutamate stimulation is similarly unperturbed by calcium depletion. Our results suggest that tastant-evoked cAMP signals are not simply a secondary consequence of calcium modulation. Instead, cAMP and released Ca²⁺ may represent independent second messenger signals downstream of taste receptors. calcium-sensitive adenylyl cyclase; capacitative entry; cross talk; taste transduction     

Membrane properties of isolated mudpuppy taste cells

The voltage-dependent currents of isolated Necturus lingual cells were studied using the whole-cell configuration of the patch-clamp technique. Nongustatory surface epithelial cells had only passive membrane properties. Small, spherical cells resembling basal cells responded to depolarizing voltage steps with predominantly outward K+ currents. Taste receptor cells generated both outward and inward currents in response to depolarizing voltage steps. Outward K+ currents activated at approximately 0 mV and increased almost linearly with increasing depolarization. The K+ current did not inactivate and was partially Ca++ dependent. One inward current activated at -40 mV, reached a peak at -20 mV, and rapidly inactivated. This transient inward current was blocked by tetrodotoxin (TTX), which indicates that it is an Na+ current. The other inward current activated at 0 mV, peaked at 30 mV, and slowly inactivated. This more sustained inward current had the kinetic and pharmacological properties of a slow Ca++ current. In addition, most taste cells had inwardly rectifying K+ currents. Sour taste stimuli (weak acids) decreased outward K+ currents and slightly reduced inward currents; bitter taste stimuli (quinine) reduced inward currents to a greater extent than outward currents. It is concluded that sour and bitter taste stimuli produce depolarizing receptor potentials, at least in part, by reducing the voltage-dependent K+ conductance.     

Caffeine inhibits cytokine expression in lymphocytes

Caffeine alters intracellular calcium signalling patterns in lymphocytes which are important for the specific regulation of activation and effector function in lymphocytes. The effect of caffeine on calcium signalling is probably mediated via a ryanodine receptor type 3 dependent intracellular calcium store which releases calcium after exposure to caffeine. Also, caffeine decreases lymphocyte cytotoxicity against allogenic myocyte. Which cytotoxic mechanisms are actually altered by caffeine is unknown. In mouse splenocyte cultures containing about 87% lymphocytes we show that concanavalin A (ConA, 5 microg/ml) stimulated cells increase the expression of TNF-alpha, IL-2 and IFN-gamma (ELISA) significantly. Caffeine (3.75 mM) inhibits cytokine expression of ConA stimulated cells almost completely. Ryanodine (1 microM) specifically blocks ryanodine receptors and thereby prevents caffeine induced calcium release. In our experiments, however, ryanodine has no effect on ConA stimulated IL-2 and IFN-gamma expression and only suppresses TNF-alpha expression by 20%. Furthermore, ryanodine does not prevent the inhibitory effect of caffeine on TNF-alpha, IL-2 and IFN-gamma expression in stimulated effector cells. We postulate that caffeine suppresses cytokine expression and thereby contributes to decreased cytotoxicity of lymphocytes against allogenic myocytes. The ryanodine receptor dependent intracellular calcium store does not seem to play a significant role in this process. Possibly, the blockade of IP3 receptors by caffeine is more important for cytokine suppression.     

Extracellular acid block and acid-enhanced inactivation of the Ca2+-activated cation channel TRPM5 involve residues in the S3-S4 and S5-S6 extracellular domains

TRPM5, a member of the superfamily of transient receptor potential ion channels, is essential for the detection of bitter, sweet, and amino acid tastes. In heterologous cell types it forms a nonselective cation channel that is activated by intracellular Ca(2+). TRPM5 is likely to be part of the taste transduction cascade, and regulators of TRPM5 are likely to affect taste sensation. In this report we show that TRPM5, but not the related channel TRPM4b, is potently blocked by extracellular acidification. External acidification has two effects, a fast reversible block of the current (IC(50) pH = 6.2) and a slower irreversible enhancement of current inactivation. Mutation of a single Glu residue in the S3-S4 linker and a His residue in the pore region each reduced sensitivity of TRPM5 currents to fast acid block (IC(50) pH = 5.8 for both), and the double mutant was nearly insensitive to acidic pH (IC(50) pH = 5.0). Prolonged exposure to acidic pH enhanced inactivation of TRPM5 currents, and mutant channels that were less sensitive to acid block were also less sensitive to acid-enhanced inactivation, suggesting an intimate association between the two processes. These processes are, however, distinct because the pore mutant H896N, which has normal sensitivity to acid block, shows significant recovery from acid-enhanced inactivation. These data show that extracellular acidification acts through specific residues on TRPM5 to block conduction through two distinct but related mechanisms and suggest a possible interaction between extracellular pH and activation and adaptation of bitter, sweet, and amino acid taste transduction.     

Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.     

Changes in cytosolic calcium monitored by inward currents during action potentials in guinea-pig ventricular cells

Action potentials were recorded from single cells isolated from guinea-pig ventricular muscle. Contraction was measured with an optical technique. Tail currents thought to be activated by cytosolic calcium were recorded when action potentials were interrupted by application of a voltage-clamp. A family of tail currents was recorded by interrupting the action potential at various times after the upstroke. The envelope of tail current amplitudes was taken as an index of changes in cytosolic calcium. Consistent with this interpretation, tail currents were negligible following intracellular loading with the calcium chelator BAPTA to suppress calcium transients. The cytosolic calcium transient estimated from the envelope of tails reached a peak approximately 50 ms after the upstroke of the action potential, and fell close to diastolic levels before repolarization was complete; 10 mM caffeine delayed the time to peak contraction, and caused a prolongation of the cytosolic calcium transient estimated from the envelope of tail currents. Caffeine also induced the appearance of a distinct late plateau phase of the action potential. Intracellular BAPTA suppressed the late plateau, contraction and tail currents in cells exposed to caffeine. Exposure to caffeine increased the time constant for decay of tail currents (from approximately 25 to 70 ms). When action potentials were greatly abbreviated by interruption with a voltage-clamp, a progressive decline occurred in the subsequent three contractions and tail currents. There was a progressive reversal of these effects over four responses when the full action potential duration was restored. None of these effects was observed in cells exposed to caffeine. Calcium-activated tail currents appear to be a useful qualitative index of changes in cytosolic calcium. The observations are consistent with the suggestion that cytosolic calcium is reduced during the plateau by a combination of calcium extrusion through Na-Ca exchange and calcium uptake into caffeine-sensitive stores. It also appears that reduction of stores loading during abbreviated action potentials reduces subsequent contraction in cells not exposed to caffeine.     

The effect of caffeine on calcium efflux and calcium translocation in skeletal and visceral muscle.

1. KCl-induced depolarization resulted in a large stimulation of the 45Ca efflux from both cockroach skeletal muscle and rat ileal smooth muscle. 2. Caffeine (10 mM) induced a large stimulation of 45Ca efflux from skeletal muscle, but a fall in the efflux from ileal muscle, especially if the efflux was previously stimulated by KCl depolarization. 3. Caffeine inhibited calcium uptake by skeletal muscle mitochondria and sarcoplasmic reticulum, was without effect on ileal muscle mitochondria, but significantly increased caclium binding by ileal muscle membrane vesicular preparations. 4. The induction of contractures and stimulation of 45Ca efflux in skeletal muscle by caffeine are clearly related to inhibition of intracellular calcium binding by the sarcoplasmic reticulum and mitochondria. 5. The relaxation of ileal muscle by caffeine and the inhibition of fibre calcium efflux correlate well with caffeine enhancement of intracellular calcium binding. These experiments suggest that the membrane vesicular compartment may be the main agency centrally involved in fibre calcium regulation in this muscle during the contraction-relaxation cycle.     

Riboflavin-binding protein is a novel bitter inhibitor

Riboflavin-binding protein (RBP) from chicken egg, which was recently reported to be a selective sweet inhibitor for protein sweeteners, was also found to be a bitter inhibitor. RBP elicited broadly tuned inhibition of various bitter substances including quinine-HCl, naringin, theobromine, caffeine, glycyl-L-phenylalanine (Gly-Phe), and denatonium benzoate, whereas several other proteins, such as ovalbumin (OVA) and beta-lactoglobulin, were ineffective in reducing bitterness of these same compounds. Both the bitter tastes of quinine and caffeine were reduced following an oral prerinse with RBP. It was found that RBP binds to quinine but not to caffeine, theobromine, naringin, and Gly-Phe. However, the binding of RBP to quinine was probably not responsible for the bitter inhibition because OVA bound to quinine as well as RBP. Based on these results, it is suggested that the bitter inhibitory effect of RBP is the consequence of its ability to interact with taste receptors rather than because it interacts with the bitter tastants themselves. RBP may have practical uses in reducing bitterness of foods and pharmaceuticals. It may also prove a useful tool in studies of mechanisms of bitter taste.     

Responses of cultured rat trigeminal ganglion neurons to bitter tastants

The initial steps in taste and olfaction result from the activation by chemical stimuli of taste receptor cells (TRCs) and olfactory receptor neurons (ORNs). In parallel with these two pathways is the chemosensitive trigeminal pathway whose neurons terminate in the oral and nasal cavities and which are activated by many of the same chemical stimuli that activate TRCs and ORNs. In a recent single unit study we investigated the responses of rat chorda tympani and glossopharnygeal neurons to a variety of bitter-tasting alkaloids, including nicotine, yohimbine, quinine, strychnine and caffeine, as well as capsaicin, the pungent ingredient in hot pepper. Here we apply many of these same compounds to cultured rat trigeminal ganglion (TG) neurons and measure changes in intracellular calcium [Ca2+]i to determine whether TG neurons will respond to these same compounds. Of the 89 neurons tested, 34% responded to 1 mM nicotine, 7% to 1 mM caffeine, 5% to 1 mM denatonium benzoate, 22% to 1 mM quinine hydrochloride, 18% to 1 mM strychnine and 55% to 1 microM capsaicin. These data suggest that neurons from the TG respond to the same bitter-tasting chemical stimuli as do TRCs and are likely to contribute information sent to the higher CNS regarding the perception of bitter/irritating chemical stimuli.      

Fast inhibition of glutamate-activated currents by caffeine

Background

Caffeine stimulates calcium-induced calcium release (CICR) in many cell types. In neurons, caffeine stimulates CICR presynaptically and thus modulates neurotransmitter release.

Methodology/Principal Findings

Using the whole-cell patch-clamp technique we found that caffeine (20 mM) reversibly increased the frequency and decreased the amplitude of miniature excitatory postsynaptic currents (mEPSCs) in neocortical neurons. The increase in mEPSC frequency is consistent with a presynaptic mechanism. Caffeine also reduced exogenously applied glutamate-activated currents, confirming a separate postsynaptic action. This inhibition developed in tens of milliseconds, consistent with block of channel currents. Caffeine (20 mM) did not reduce currents activated by exogenous NMDA, indicating that caffeine block is specific to non-NMDA type glutamate receptors.

Conclusions/Significance

Caffeine-induced inhibition of mEPSC amplitude occurs through postsynaptic block of non-NMDA type ionotropic glutamate receptors. Caffeine thus has both pre and postsynaptic sites of action at excitatory synapses.     

Direct measurement of translingual epithelial NaCl and KCl currents during the chorda tympani taste response.

We have measured the NaCl or KCl currents under voltage clamp across the dorsal lingual epithelium of the rat and simultaneously the response of the taste nerves. Under short-circuit conditions a NaCl stimulus evoked an inward current (first current) that coincided with excitation of the chorda tympani. This was followed by a slower inward current (second current) that matched the kinetics of taste nerve adaptation. The peak first current and the coincident neural response satisfied the same saturating NaCl concentration dependence. Both first and second currents were partially blocked by amiloride as were the phasic and tonic components of the neural response. The NaCl-evoked second current was completely blocked by ouabain. Investigation of the NaCl-evoked current and the neural response over a range of clamped voltages showed that inward negative potentials enhanced the inward current and the neural response to 0.3 M NaCl. Sufficiently high inward positive potentials reversed the current, and made the neural response independent of further changes in voltage. Therefore, one of the NaCl taste transduction mechanisms is voltage dependent while the other is voltage independent. A KCl stimulus also evoked an inward short-circuit current, but this and the neural response were not amiloride-sensitive. The data indicate that neural adaptation to a NaCl stimulus, but not a KCl stimulus, is mediated by cell Na/K pumps. A model is proposed in which the connection between the NaCl-evoked second current and cell repolarization is demonstrated.     

Iontophoretic application of bitter taste stimuli in hamsters

Recent electrophysiological studies on the iontophoretic applicationof taste stimuli by weak electric currents using rodents andfrogs have produced stimuli which appear to mimic the actionof salty, sour and sweet solutions. However, there has beenno report of an ionic stimulus which might serve as a bitteriontophoretic probe. Many common bitter stimuli are either uncharged(e.g. quinine, urea) or have mixed quality sensations (e.g.the bitter salts KCl, MgCl₂) and therefore are unsuitable. Thisreport investigates the use of four organic anions, all of whichare bitter to humans, which may serve as potential bitter stimulifor iontophoretic application to the tongue of the hamster whilerecording electrophysiologically from its chorda tympani nerve.These anions are m-nitrobenzene sulfonate (NBSA), picrate, cholateand m-nitrobenzoate (NBA). The electrophysiological responsesto cathodal polarization via these four anions plus saccharin,an effective cathodal stimulus in the hamster, form the sameefficacy series as chemical (i.e. normal sapid) presentationsof sodium salts of these anions, i.e. saccharin > NBSA >picrate > NBA > cholate. Behavioral evidence suggeststhat NBSA is sweet to hamsters while the latter three anions,picrate, NBA and cholate, are bitter. Electrophyiological observations,based on magnitude of response, appear to support these behavioralfindings. It was concluded that picrate, NBA and cholate mayserve as useful bitter stimulus probes for ionto-phoretic applicationin the hamster.     

SH—SY5Y细胞的钙缓冲研究

目的：研究SH－SY5Y神经杂交瘤细胞的钙缓冲能力。方法：通过膜片钳手段,测量未分化的SH－SY5Y细胞钙离子通道电流;并应用显微荧光测量游离钙离子浓度和高钾去极化的方法,研究胞内Ca^2 浓度上升后浓度恢复的动力学过程。结果：未分化的SH－SY5Y细胞存在钙离子通道电流,在刺激时间间隔较短时（＜150s）,胞内钙浓度的恢复过程会由于缓冲机制的饱和而变慢;而时间间隔＞150s时,缓冲物质则可以基本恢复使得胞内钙的恢复过程基本保持不变。结论：钙缓冲蛋白在细胞内钙浓度的调节中起重要作用。     

THE IMPORTANCE OF CAFFEINE AS A FLAVOR COMPONENT IN BEVERAGES

Caffeine contributes to the flavor profiles of beverage and food products, and other bitter compounds, such as quinine, cannot replicate caffeine's bitterness. Research also has indicated caffeine's ability to enhance desirable tastes, such as sweetness and saltiness, in beverages by modifying neurological pathways. Caffeine may reduce potential " off-flavors" by solubilizing some compounds, while having little effect on other compounds considered important in beverage flavor profiles. Exposure to caffeine may enhance cognition and potentially heighten the capacity for smell and taste. Thus, use of caffeine in beverages may positively affect flavor profiles and the experience of beverage consumption.     

Analysis of calcium activated chloride current fluctuations in Xenopus laevis oocytes.

Fluctuations of calcium activated chloride currents were investigated in oocytes of Xenopus laevis. The method of noise analysis and the model of chloride channels activation by calcium ions were used to estimate the chloride channels lifetime and the average frequency of current fluctuations, which depends on changes of cytoplasmic calcium concentration. This current fluctuations can be evoked by activation of cholinergic receptors or inhibition by Na3VO4 of plasma membrane Ca(2+)-ATPase. The average opening lifetime of chloride channels was approximately 100 ms. The frequency of fluctuations increased with the increasing extracellular calcium concentrations and external ACh concentrations. Caffeine in 2 mmol/l concentration changed the current fluctuations into oscillations with a period of about 18-20s. Ten mmol/l caffeine fully inhibited the oscillation activity.     

Bitter taste stimuli induce differential neural codes in mouse brain

A growing literature suggests taste stimuli commonly classified as "bitter" induce heterogeneous neural and perceptual responses. Here, the central processing of bitter stimuli was studied in mice with genetically controlled bitter taste profiles. Using these mice removed genetic heterogeneity as a factor influencing gustatory neural codes for bitter stimuli. Electrophysiological activity (spikes) was recorded from single neurons in the nucleus tractus solitarius during oral delivery of taste solutions (26 total), including concentration series of the bitter tastants quinine, denatonium benzoate, cycloheximide, and sucrose octaacetate (SOA), presented to the whole mouth for 5 s. Seventy-nine neurons were sampled; in many cases multiple cells (2 to 5) were recorded from a mouse. Results showed bitter stimuli induced variable gustatory activity. For example, although some neurons responded robustly to quinine and cycloheximide, others displayed concentration-dependent activity (p<0.05) to quinine but not cycloheximide. Differential activity to bitter stimuli was observed across multiple neurons recorded from one animal in several mice. Across all cells, quinine and denatonium induced correlated spatial responses that differed (p<0.05) from those to cycloheximide and SOA. Modeling spatiotemporal neural ensemble activity revealed responses to quinine/denatonium and cycloheximide/SOA diverged during only an early, at least 1 s wide period of the taste response. Our findings highlight how temporal features of sensory processing contribute differences among bitter taste codes and build on data suggesting heterogeneity among "bitter" stimuli, data that challenge a strict monoguesia model for the bitter quality.     

Rapid entry of bitter and sweet tastants into liposomes and taste cells: implications for signal transduction

Someamphipathic bitter tastants and non-sugar sweeteners are directactivators of G proteins and stimulate transduction pathways in cellsnot related to taste. We demonstrate that the amphipathic bittertastants quinine and cyclo(Leu-Trp) and the non-sugar sweetener saccharin translocate rapidly through multilamellar liposomes. Furthermore, when rat circumvallate (CV) taste buds were incubated withthe above tastants for 30 s, their intracellular concentrations increased by 3.5- to 7-fold relative to their extracellularconcentrations. The time course of this dramatic accumulation was alsomonitored in situ in rat single CV taste buds under a confocallaser-scanning microscope. Tastants were clearly localized to the tastecell cytosol. It is proposed that, due to their rapid permeation into taste cells, these amphipathic tastants may be available for activation of signal transduction components (e.g., G proteins) directly withinthe time course of taste sensation. Such activation may occur inaddition to the action of these tastants on putative G protein-coupledreceptors. This phenomenon may be related to the slow taste onset andlingering aftertaste typically produced by many bitter tastants andnon-sugar sweeteners.