Bitter Taste Transduction of Denatonium in the MudpuppyNecturus maculosus

Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado 80523, USA.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.75). 06/1997; 17(10):3580-7.
Source: PubMed

ABSTRACT Bitter substances are a structurally diverse group of compounds that appear to act via several transduction mechanisms. The bitter-tasting denatonium ion has been proposed to act via two different G-protein-regulated pathways, one involving inositol 1,4, 5-trisphosphate and raised intracellular calcium levels, the other involving phosphodiesterase and membrane depolarization via a cyclic nucleotide-suppressible cation channel. The aim of the present study was to examine these transduction mechanisms in taste cells of the mudpuppy Necturus maculosus by calcium-imaging and whole-cell recording. Denatonium benzoate increased intracellular calcium levels and induced an outward current independently of extracellular calcium. The denatonium-induced increase in intracellular calcium was inhibited by U73122, an inhibitor of phospholipase C, and by thapsigargin, an inhibitor of calcium transport into intracellular stores. The denatonium-induced outward current was blocked by GDP-beta-S, a blocker of G-protein activation. Neither resting nor denatonium-induced intracellular calcium levels were affected by inhibition of phosphodiesterase (with IBMX) or adenylate cyclase (with SQ22536) or by raising intracellular cyclic nucleotides directly (with cell permeant analogs). Our results support the hypothesis that denatonium is transduced via a G-protein cascade involving phospholipase C, inositol 1,4,5-trisphosphate, and raised intracellular calcium levels. Our results do not support the hypothesis that denatonium is transduced via phosphodiesterase and cAMP.

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Available from: Sue Kinnamon, Nov 20, 2014
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    • "Both second messenger IP 3 and DAG produced as the consequence of PLC activation are capable of increasing intracellular Ca 2+ via either internal or external sources in sensory receptor cells (Restrepo et al., 1990; Schild et al., 1995; Ogura et al., 1997; Lucas et al., 2003; Zhang et al., 2010). We removed extracellular Ca 2+ from the bath solution to determine whether the external or internal Ca 2+ source is responsible for the PLC-induced Ca 2+ increases. "
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    ABSTRACT: Phospholipase C (PLC) and internal Ca(2+) stores are involved in a variety of cellular functions. However, our understanding of PLC in mammalian olfactory sensory neurons (OSNs) is generally limited to its controversial role in odor transduction. Here we employed single-cell Ca(2+) imaging and molecular approaches to investigate PLC-mediated Ca(2+) responses and its isozyme gene transcript expression. We found that the pan-PLC activator m-3M3FBS (25 μM) induces intracellular Ca(2+) increases in vast majority of isolated mouse OSNs tested. Both the response amplitude and percent responding cells depend on m-3M3FBS concentrations. In contrast, the inactive analog o-3M3FBS fails to induce Ca(2+) responses. The m-3M3FBS-induced Ca(2+) increase is blocked by the PLC inhibitor U73122, while its inactive analog U73433 has no effect. Removal of extracellular Ca(2+) does not change significantly the m-3M3FBS-induced Ca(2+) response amplitude. Additionally, in the absence of external Ca(2+), we found that a subset of OSNs respond to an odorant mixture with small Ca(2+) increases, which are significantly suppressed by U73122. Furthermore, using reverse transcription polymerase chain reaction and real-time quantitative polymerase chain reaction, we found that multiple PLC isozyme gene transcripts are expressed in olfactory turbinate tissue in various levels. Using RNA in situ hybridization analysis, we further show expression of β4, γ1, γ2 gene transcripts in OSNs. Taken together, our results establish that PLC isozymes are potent enzymes for mobilizing intracellular Ca(2+) in mouse OSNs and provide molecular insight for PLC isozymes-mediated complex cell signaling and regulation in the peripheral olfactory epithelium.
    Frontiers in Cellular Neuroscience 10/2014; 8:336. DOI:10.3389/fncel.2014.00336 · 4.18 Impact Factor
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    • "For that reason, taste cells with a resting baseline calcium level greater than 200 nM were not used in these studies to ensure that we were not including damaged cells in our analyses (Hacker and Medler 2008; Laskowski and Medler 2009; Szebenyi et al. 2010). This value was based on the fact that most neurons have a resting calcium value around 100 nM and taste cells from other studies have reported similar values (Ogura et al. 1997; Baryshnikov et al. 2003). "
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    ABSTRACT: Peripheral taste receptor cells depend on distinct calcium signals to generate appropriate cellular responses that relay taste information to the central nervous system. Some taste cells have conventional chemical synapses and rely on calcium influx through voltage-gated calcium channels. Other taste cells lack these synapses and depend on calcium release from stores to formulate an output signal through a hemichannel. Despite the importance of calcium signaling in taste cells, little is known about how these signals are regulated. This review summarizes recent studies that have identified 2 calcium clearance mechanisms expressed in taste cells, including mitochondrial calcium uptake and sodium/calcium exchangers (NCXs). These studies identified a unique constitutive calcium influx that contributes to maintaining appropriate calcium homeostasis in taste cells and the role of the mitochondria and exchangers in this process. The additional role of NCXs in the regulation of evoked calcium responses is also discussed. Clearly, calcium signaling is a dynamic process in taste cells and appears to be more complex than has previously been appreciated.
    Chemical Senses 11/2010; 35(9):753-65. DOI:10.1093/chemse/bjq082 · 3.28 Impact Factor
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    • "We used the same analysis paradigm to measure the effect of inhibiting NCXs on the denatonium-evoked calcium response (Fig. 2). Denatonium was used as a representative bitter stimulus which activates GPCRs to cause calcium release from internal stores (Akabas et al. 1988; Margolskee 2002; Ogura et al. 1997; Zhang et al. 2003). We chose to use 10 mM denatonium to maximally activate the evoked calcium response. "
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    ABSTRACT: Taste cells use multiple signaling mechanisms to generate appropriate cellular responses to discrete taste stimuli. Some taste stimuli activate G protein coupled receptors (GPCRs) that cause calcium release from intracellular stores while other stimuli depolarize taste cells to cause calcium influx through voltage-gated calcium channels (VGCCs). While the signaling mechanisms that initiate calcium signals have been described in taste cells, the calcium clearance mechanisms (CCMs) that contribute to the termination of these signals have not been identified. In this study, we used calcium imaging to define the role of sodium-calcium exchangers (NCXs) in the termination of evoked calcium responses. We found that NCXs regulate the calcium signals that rely on calcium influx at the plasma membrane but do not significantly contribute to the calcium signals that depend on calcium release from internal stores. Our data indicate that this selective regulation of calcium signals by NCXs is due primarily to their location in the cell rather than to the differences in cytosolic calcium loads. This is the first report to define the physiological role for any of the CCMs utilized by taste cells to regulate their evoked calcium responses.
    Journal of Neurophysiology 05/2010; 104(1):529-38. DOI:10.1152/jn.00118.2010 · 3.04 Impact Factor
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