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: 6.34). 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 · 4.29 Impact Factor
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    • "T2Rs belong to the family of G protein coupled receptors(GPCRs) and activate phospholipase C (PLCβ2), to produce inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) [22]. IP3 binds to IP3 receptors and elicits a release of Ca2+ from the sarcoplasmic reticulum (SR) [23]. Moreover, taste receptor cell responses to the bitter stimulus, denatonium, involve Ca2+ influx via store-operated channels [24]. "
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    ABSTRACT: Chloroquine (CQ), a bitter tasting drug widely used in treatment of malaria, is associated gastrointestinal side effects including nausea or diarrhea. In the present study, we investigated the effect of CQ on electrolyte transport in rat ileum using the Ussing chamber technique. The results showed that CQ evoked an increase in short circuit current (ISC ) in rat ileum at lower concentration (≤5×10(-4) M ) but induced a decrease at higher concentrations (≥10(-3) M). These responses were not affected by tetrodotoxin (TTX). Other bitter compounds, such as denatoniumbenzoate and quinine, exhibited similar effects. CQ-evoked increase in ISC was partly reduced by amiloride(10(-4) M), a blocker of epithelial Na(+) channels. Furosemide (10(-4) M), an inhibitor of Na(+)-K(+) -2Cl(-) co-transporter, also inhibited the increased ISC response to CQ, whereas another Cl(-) channel inhibitor, CFTR(inh)-172(10(-5)M), had no effect. Intriguingly, CQ-evoked increases were almost completely abolished by niflumic acid (10(-4)M), a relatively specific Ca(2+)-activated Cl(-) channel (CaCC) inhibitor. Furthermore, other CaCC inhibitors, such as DIDS and NPPB, also exhibited similar effects. CQ-induced increases in ISC were also abolished by thapsigargin(10(-6)M), a Ca(2+) pump inhibitor and in the absence of either Cl(-) or Ca(2+) from bathing solutions. Further studies demonstrated that T2R and CaCC-TMEM16A were colocalized in small intestinal epithelial cells and the T2R agonist CQ evoked an increase of intracelluar Ca(2+) in small intestinal epithelial cells. Taken together, these results demonstrate that CQ induces Cl (-) secretion in rat ileum through CaCC at low concentrations, suggesting a novel explanation for CQ-associated gastrointestinal side-effects during the treatment of malaria.
    PLoS ONE 01/2014; 9(1):e87627. DOI:10.1371/journal.pone.0087627 · 3.23 Impact Factor
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    • "From these results (Fig. S3) the best working antiserum combination was chosen to perform the experiment on human circumvallate papillae sections. We performed double-immunofluorescence labeling experiments using the anti-TAS2R38 antiserum in combination with a PLCβ2 antiserum to demonstrate expression of TAS2R38 in type II taste receptor cells [22]–[24]. As seen in figure 5, both antisera label subsets of cells within taste buds of human circumvallate papillae. "
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    ABSTRACT: The sense of taste plays an important role in the evaluation of the nutrient composition of consumed food. Bitter taste in particular is believed to serve a warning function against the ingestion of poisonous substances. In the past years enormous progress was made in the characterization of bitter taste receptors, including their gene expression patterns, pharmacological features and presumed physiological roles in gustatory as well as in non-gustatory tissues. However, due to a lack in TAS2R-specifc antibodies the localization of receptor proteins within gustatory tissues has never been analyzed. In the present study we have screened a panel of commercially available antisera raised against human bitter taste receptors by immunocytochemical experiments. One of these antisera was found to be highly specific for the human bitter taste receptor TAS2R38. We further demonstrate that this antibody is able to detect heterologously expressed TAS2R38 protein on Western blots. The antiserum is, however, not able to interfere significantly with TAS2R38 function in cell based calcium imaging analyses. Most importantly, we were able to demonstrate the presence of TAS2R38 protein in human gustatory papillae. Using double immunofluorescence we show that TAS2R38-positive cells form a subpopulation of PLCbeta2 expressing cells. On a subcellular level the localization of this bitter taste receptor is neither restricted to the cell surface nor particularly enriched at the level of the microvilli protruding into the pore region of the taste buds, but rather evenly distributed over the entire cell body.
    PLoS ONE 07/2012; 7(7):e40304. DOI:10.1371/journal.pone.0040304 · 3.23 Impact Factor
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