Gustducin is a transducin-like G protein selectively expressed in taste receptor cells. The alpha subunit of gustducin (alpha-gustducin) is critical for transduction of responses to bitter or sweet compounds. We identified a G-protein gamma subunit (Ggamma13) that colocalized with alpha-gustducin in taste receptor cells. Of 19 alpha-gustducin/Ggamma13-positive taste receptor cells profiled, all expressed the G protein beta3 subunit (Gbeta3); approximately 80% also expressed Gbeta1. Gustducin heterotrimers (alpha-gustducin/Gbeta1/Ggamma13) were activated by taste cell membranes plus bitter denatonium. Antibodies against Ggamma13 blocked the denatonium-induced increase of inositol trisphosphate (IP3) in taste tissue. We conclude that gustducin heterotrimers transduce responses to bitter and sweet compounds via alpha-gustducin's regulation of phosphodiesterase (PDE) and Gbetagamma's activation of phospholipase C (PLC).

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.

Taste is the least understood among sensory systems, and bitter taste mechanisms pose a special challenge because they are elicited by a large variety of compounds. We studied bitter taste signal transduction with the quench-flow method and monitored the rapid kinetics of the second messenger guanosine 3',5'-cyclic monophosphate (cGMP) production and degradation in mouse taste tissue. In response to the bitter stimulants, caffeine and theophylline but not strychnine or denatonium cGMP levels demonstrated a rapid and transient increase that peaked at 50 ms and gradually declined throughout the following 4.5 s. The theophylline- and caffeine-induced effect was rapid, transient, concentration dependent and gustatory tissue-specific. The effect could be partially suppressed in the presence of the soluble guanylyl cyclase (GC) inhibitor 10 microM ODQ and 30 microM methylene blue but not 50 microM LY 83583 and boosted by nitric oxide donors 25 microM NOR-3 or 100 microM sodium nitroprusside. The proposed mechanism for this novel cGMP-mediated bitter taste signal transduction is cGMP production partially by the soluble GC and caffeine-induced inhibition of one or several phosphodiesterases.

Ceramide, a product of sphingomyelin turnover, is a lipid second messenger that mediates diverse signaling pathways, including those leading to cell cycle arrest and differentiation. The mechanism(s) by which ceramide signals downstream events have not been fully elucidated. Here we show that, in Xenopus laevis oocytes, ceramide-induced maturation is associated with the release of intracellular calcium stores. Ceramide caused a dose-dependent elevation in the second messenger inositol 1,4,5-trisphosphate (IP3) via activation of Gq/11α and phospholipase C-βX. Elevation of IP3, in turn, activated the IP3 receptor calcium release channel on the endoplasmic reticulum, resulting in a rise in cytoplasmic calcium. Thus our study demonstrates that cross talk between the ceramide and phosphoinositide signaling pathways modulates intracellular calcium homeostasis.

A B S T R A C TAiJ mice were subjected to restraint and/or food and water deprivation for 24 or 48 hours beginning on day 14 of' pregnancy. The 48-hour treatment sharply increascd the cleft palate and resorption rates. The data strongly suggest that restraint can cause cleft palate if imposed for an extended period of time.