cyclic-gmp and denatonium-benzoate

Current evidence points to the existence of multiple processes for bitter taste transduction. Previous work demonstrated involvement of the polyphosphoinositide system and an alpha-gustducin (Galpha(gust))-mediated stimulation of phosphodiesterase in bitter taste transduction. Additionally, a taste-enriched G protein gamma-subunit, Ggamma(13), colocalizes with Galpha(gust) and mediates the denatonium-stimulated production of inositol 1,4,5-trisphosphate (IP(3)). Using quench-flow techniques, we show here that the bitter stimuli, denatonium and strychnine, induce rapid (50-100 ms) and transient reductions in cAMP and cGMP and increases in IP(3) in murine taste tissue. This decrease of cyclic nucleotides is inhibited by Galpha(gust) antibodies, whereas the increase in IP(3) is not affected by antibodies to Galpha(gust). IP(3) production is inhibited by antibodies specific to phospholipase C-beta(2) (PLC-beta(2)), a PLC isoform known to be activated by Gbetagamma-subunits. Antibodies to PLC-beta(3) or to PLC-beta(4) were without effect. These data suggest a transduction mechanism for bitter taste involving the rapid and transient metabolism of dual second messenger systems, both mediated through a taste cell G protein, likely composed of Galpha(gust)/beta/gamma(13), with both systems being simultaneously activated in the same bitter-sensitive taste receptor cell.

Topics: Animals; Cyclic AMP; Cyclic GMP; Glycine Agents; Inositol 1,4,5-Trisphosphate; Isoenzymes; Mice; Mice, Inbred Strains; Phospholipase C beta; Quaternary Ammonium Compounds; Second Messenger Systems; Signal Transduction; Strychnine; Taste; Taste Buds; Transducin; Type C Phospholipases

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.

Topics: 1-Methyl-3-isobutylxanthine; Adenine; Adenylate Cyclase Toxin; Animals; Calcium; Calcium Channels; Cyclic AMP; Cyclic GMP; Enzyme Inhibitors; Estrenes; Fluorescent Dyes; Fura-2; GTP-Binding Proteins; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Membrane Potentials; Necturus; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Pyrrolidinones; Quaternary Ammonium Compounds; Receptors, Cytoplasmic and Nuclear; Ryanodine; Signal Transduction; Taste; Taste Buds; Thapsigargin; Virulence Factors, Bordetella