The taste-selective G protein, a-gustducin (a-gus) is homologous to a-transducin and activates phosphodiesterase (PDE) in vitro. a-Gus-knockout mice are compromized to bitter, sweet and umami taste stimuli, suggesting a central role in taste transduction. Here, we suggest a different role for Ga-gus. In taste buds of a-gus-knockout mice, basal (unstimulated) cAMP levels are high compared to those of wild-type mice. Further, H-89, a cAMP-dependent protein kinase inhibitor, dramatically unmasks responses to the bitter tastant denatonium in gus-lineage cells of knockout mice. We propose that an important role of a-gus is to maintain cAMP levels tonically low to ensure adequate Ca 2+ signaling.

We previously showed that rat taste buds express several adenylyl cyclases (ACs) of which only AC8 is known to be stimulated by Ca 2ϩ . Here we demonstrate by direct measurements of cAMP levels that AC activity in taste buds is stimulated by treatments that elevate intracellular Ca 2ϩ . Specifically, 5 M thapsigargin or 3 M A-23187 (calcium ionophore), both of which increase intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i), lead to a significant elevation of cAMP levels. This calcium stimulation of AC activity requires extracellular Ca 2ϩ , suggesting that it is dependent on Ca 2ϩ 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 C␤2 (i.e., cells that elevate [Ca 2ϩ ]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 Ca 2ϩ may represent independent second messenger signals downstream of taste receptors.calcium-sensitive adenylyl cyclase; capacitative entry; cross talk; taste transduction DURING THE PAST DECADE, numerous advances have been made in our understanding of taste transduction mechanisms. Most tastants of the sweet, bitter, and umami classes are thought to activate G protein-coupled taste receptors and their heterotrimeric G proteins. The G␥ 13 subunit appears to be taste specific and associated with G␤ 1 (14). This G␤ 1 ␥ 13 dimer has been shown directly to activate a phospholipase C (PLC␤ 2 ), stimulating the production of inositol 1,4,5-trisphosphate (IP 3 ) and eventually triggering an increase in cytoplasmic Ca 2ϩ (14,23,35,38). Strong support for this sequence of signaling events is derived from the pronounced taste deficit that results from genetic ablation of PLC␤ 2 (45). Although release of stored Ca 2ϩ (triggered by PLC␤ 2 activity) is essential, taste-evoked calcium transients in taste cells may also include a component of capacitative entry from the extracellular medium (28). Despite the recent emphasis on this IP 3 -mediated calcium release pathway, there is evidence that changes in cAMP and cGMP concentration occur after tastant stimulation. Cyclic nucleotide modulation has been demonstrated after stimulation with some sweet, bitter, and umami stimuli (1,39,44), and exogenously applied cAMP appears to mimic tastant-evoked activity (8). Nevertheless, the significance and source of cAMP and cGMP in taste transduction are unclear, given the essential role of...

Abaffy, Tatjana, Kristina R. Trubey, and Nirupa Chaudhari. Adenylyl cyclase expression and modulation of cAMP in rat taste cells. Am J Physiol Cell Physiol 284: C1420-C1428, 2003. First published February 26, 2003 10.1152/ajpcell.00556.2002cAMP is a second messenger implicated in sensory transduction for taste. The identity of adenylyl cyclase (AC) in taste cells has not been explored. We have employed RT-PCR to identify the AC isoforms present in taste cells and found that AC 4, 6, and 8 are expressed as mRNAs in taste tissue. These proteins are also expressed in a subset of taste cells as revealed by immunohistochemistry. Alterations of cAMP concentrations are associated with transduction of taste stimuli of several classes. The involvement of particular ACs in this modulation has not been investigated. We demonstrate that glutamate, which is a potent stimulus eliciting a taste quality termed umami, causes a decrease in cAMP in forskolin-treated taste cells. The potentiation of this response by inosine monophosphate, the lack of response to D-glutamate, and the lack of response to umami stimuli in nonsensory lingual epithelium all suggest that the cAMP modulation represents umami taste transduction. Because cAMP downregulation via ACs can be mediated through G␣ i proteins, we examined the colocalization of the detected ACs with G␣i proteins and found that 66% of AC8 immunopositive taste cells are also positive for gustducin, a taste-specific G␣ i protein. Whether AC8 is directly involved in signal transduction of umami taste remains to be established. immunohistochemistry; glutamate; umami; taste transduction; gustducin MEMBRANE-ASSOCIATED ADENYLYL CYCLASES (AC) are enzymes that are central in the regulation of intracellular cAMP levels. ACs, comprising a family of nine isoforms (AC1-9), are integral membrane proteins, composed of two cytosolic catalytic domains, C1 and C2, each preceded by six transmembrane domains (16, 34). The catalytic domains are highly conserved across all ACs and even across many guanylate cyclases and are responsible for the synthesis of cAMP using ATP as a substrate. Physiologically, G protein-coupled receptors (GPCRs) regulate ACs through heterotrimeric G protein subunits, specifically the stimulatory G␣ s , the inhibitory G␣ i , and/or certain G␤␥-subunits (9, 45). All AC isoforms can be stimulated by G␣ s and by forskolin (FSK), a diterpene commonly used in cAMP analysis.Inhibition by G␣ i is limited to a subset of AC isoforms. Inhibitory G␣ i subunits are able to overcome the FSKmediated activation of AC1, 3, 5, 6, 8, and 9, presumably because FSK binds near the catalytic site, distant from the G␣ i binding site (16,55). ACs also differ in their response to modulators such as Ca 2ϩ and calmodulin. Indeed, a unified classification of ACs incorporates both such regulatory properties and their sequence similarities.Taste buds are heterogeneous clusters of taste receptor cells, each of which responds to subsets of taste stimuli and contains distinct populations of cell signaling mole...

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