Taste receptor cells are specialized epithelial cells, which are organized into discrete endorgans called taste buds. Typical taste buds contain 50-100 polarized taste cells, which extend from the basal lamina to the taste pore, where apical microvilli protrude into the oral cavity. The basolateral membrane forms chemical synapses with primary gustatory neurons (Fig. 1A). In mammals, lingual taste buds are housed in connective tissue structures called papillae. Fungiform papillae are located on the anterior two-thirds of the tongue and typically contain 1-2 taste buds each. Vallate and foliate papillae are found on the posterior tongue and house several hundred taste buds each. Taste transduction begins when sapid stimuli interact with the apical membrane of taste cells, usually resulting in taste cell depolarization, calcium influx, and transmitter release onto gustatory afferent neurons. Simple stimuli, such as salts and acids depolarize taste cells by direct interaction with apical ion channels. In contrast, complex stimuli, such as sugars, amino acids, and most bitter compounds bind to G protein coupled receptors, initiating intracellular signaling cascades that culminate in Ca²⁺ influx or release of Ca²⁺ from intracellular stores 123.

Inositol 1,4,5-trisphosphate (IP₃) is an important second messenger in both bitter and sweet taste transduction. In both pathways, activation of taste receptors stimulates a G protein-coupled cascade resulting in activation of phospholipase C (PLC), which cleaves phosphoinositol bisphosphate (PIP₂) to produce the second messengers IP₃ and diacylglycerol (DAG). The soluble messenger IP₃ binds to receptors located on calcium store membranes, causing release of calcium into the cytosol, while DAG remains in the membrane, where it can activate downstream effectors. While little is known about the role of IP₃ in sweet taste transduction, considerable data indicate that IP₃ plays an important role in bitter transduction. The first evidence for the involvement of IP₃ in bitter transduction was obtained by Akabas et al. [4], who used Ca²⁺ imaging to show that the bitter stimulus denatonium causes release of Ca²⁺ from intracellular stores. More recently, biochemical measurements have shown that several bitter compounds elevate IP₃ in taste tissue 5678.

Other studies, however, suggested that a decrease in cAMP, rather than an increase in IP₃, mediated bitter transduction. In 1992, a chemosensory specific G protein, α-gustducin, was identified in a subset of taste cells [9]. Alpha-gustducin, which is closely related to the rod and cone transducins, activates PDE to reduce intracellular levels of cAMP [13]. Evidence for gustducin's role in bitter taste came from knockout studies, in which a targeted deletion of α-gustducin resulted in mice with a reduced sensitivity for bitter compounds [10]. More recently, a variety of bitter compounds have been shown to activate gustducin in biochemical assays [11, 12], causing a decrease in intracellular cAMP levels via activation of PDE [13]. It is now known that individual bitter receptors modulate both the IP₃ and cAMP pathways (Fig. 1B). Bitter compounds bind to a family of G protein-coupled receptors called the T2R [14, 15] or TRB [16] receptors, which activate a heterotrimeric G protein consisting of α-gustducin and a βγ complex containing β3 [17] and γ13 [8]. Alpha-gustducin activates PDE to decrease intracellular levels of cAMP [13], while its βγ partners stimulate PLCβ2 to produce IP₃ and DAG [18].

Although it is clear that IP₃ binds to receptors located on intracellular Ca²⁺ stores, the specific identity of IP₃ receptors in taste cells is not known. There are at least 3 known isotypes of IP₃ receptors encoded by different genes [19]. Each protein product is about 300 Kda. Four subunits assemble to form a functional channel. Both homomultimeres and heteromultimeres have been reported [19]. The N terminus of each subunit houses the IP₃ binding domain while the C terminus anchors the protein to the membrane, is involved in the formation of the tetrameric protein, and forms the Ca²⁺ pore region. The general structure of each isoform is similar, however they differ in primary sequence, distribution, regulation, and IP₃ affinity [19].

In this study we used immunocytochemical methods to determine which IP₃R isoform is expressed in taste cells and to examine the expression patterns of IP₃ receptors relative to other proteins known to be important for taste transduction. We report that the Type III IP₃ receptor is the dominant isotype expressed in rodent taste cells and that it is primarily found in the same subset of taste cells as other known signaling components of bitter transduction. A preliminary account of this work was published in abstract form [20].

IP₃R3 is heavily expressed in a large subset of vallate taste cells of both mouse and rat, suggesting that IP₃R3 plays a similar role in both species. IP₃R3 appears to be located throughout the cytoplasm of taste cells, consistent with its expected location on the smooth endoplasmic membrane [21]. In other cells, IP₃ receptors have also been found on the plasma membrane [19], but because of heavy cytoplasmic labeling, we were unable to resolve whether it was also located on the plasma membrane. One caveat is that antigen retrieval was necessary to observe IP₃R3 labeling. However, using this method with α-gustducin, PLCβ2, and γ13 antibodies did not alter their immunoreactivities; similar results were obtained with and without antigen retrieval. Thus, we do not believe that antigen retrieval compromised our interpretation of the results.

Taste cells expressing IP₃R3 have an elongate, bipolar morphology, suggestive of Type II taste cells [22]. Indeed, a subset of the IP₃R3 immunoreactive taste cells is also immunoreactive for α-gustducin, which has been identified exclusively in Type II cells [23]. However, whether IP₃R3 is expressed exclusively in Type II cells awaits further investigation. It is noteworthy that a subset of taste cells does not express IP₃ receptors. This raises the question as to whether these cells have intracellular Ca²⁺ release mechanisms. Ryanodine receptors also mediate release of Ca²⁺ from intracellular stores, however a previous study showed no effect of ryanodine on bitter taste responses in Necturus taste cells [24].

IP₃R3 immunoreactivity was expressed in the same subset of taste cells as PLCβ2 and γ13, and by inference from other data, β3 [8]. Antibodies against these proteins have been shown to inhibit IP₃ formation to bitter compounds in taste cells [8, 17, 18], suggesting that they are important components of the bitter-stimulated IP₃ pathway. It is of interest that only a subset of IP₃R3 IR cells express α-gustducin, a G protein known to be involved with bitter transduction. These data suggest that alpha subunit(s) in addition to α-gustducin must be involved with the IP₃ signaling pathway in α-gustducin negative cells. Several G protein alpha subunits have been identified in taste cells, and are potential candidates for this role. These include α-transducin [25], Gαi-2, Gαi-3, Gαs [26], and Gα15 and Gαq [27]. Further experiments will be required to identify the additional alpha subunits that couple to this pathway, and the receptors that activate these G proteins.

In addition to its role in bitter transduction, IP₃ is involved in the transduction of artificial sweeteners [28]. Interestingly, mice lacking α-gustducin are compromised in the detection of sweet compounds as well as bitter compounds, suggesting that sweet receptors may also couple to α-gustducin [10]. Approximately two-thirds of the α-gustducin positive vallate taste cells express T2R/TRB receptors [15]. It is possible that the remaining α-gustducin positive vallate taste cells express receptors for synthetic sweeteners, and that they couple to the IP₃ signaling pathway. Thus, IP₃R3 may be involved with sweet as well as bitter taste transduction.

IP₃R3 is widely expressed in cells in a variety of tissues including adult pancreatic islets, kidney, gastrointestinal tract, salivary glands, and brain [29, 30]. Many of these cell types, including taste cells, are polarized, where Ca²⁺ signals are initiated on the apical membrane and must propagate long intracellular distances. IP₃R3 is particularly well suited for this function, since it is the only IP₃ receptor isotype that is not inhibited at high Ca²⁺ concentrations [31]. In fact, under certain conditions, Ca²⁺ can stimulate IP₃R3, making it a likely candidate for participation in the propagation of Ca²⁺ oscillations. In taste cells Ca²⁺ oscillations have been observed in response to bitter stimuli (T. Ogura and S.C. Kinnamon, unpub. observations), and it's likely that IP₃R3 participates in these Ca²⁺ oscillations.

Another interesting feature of IP₃R3 is that cAMP-dependent phosphorylation can inhibit its activity in pancreatic acinar cells [32, 33]. In these cells, cAMP-dependent phosphorylation decreases Ca²⁺ release from intracellular stores and slows the frequency of Ca²⁺ oscillations. These data suggest a possible role for α-gustducin in bitter taste transduction. Specifically, activation of α-gustducin, which decreases intracellular cAMP by activation of PDE [11], may lead to a decrease in the cAMP-dependent phosphorylation of IP₃R3. This would disrupt the negative control of the receptor and potentiate the Ca²⁺ response. Interestingly, Gαi-2, another alpha subunit heavily expressed in taste cells [26], also functions to decrease intracellular levels of cAMP and may lead to regulation of IP₃R3. Further experiments will be necessary to clarify the role of these alpha subunits in regulation of the IP₃ pathway in taste cells.

The principal finding in this study is the identification of IP₃R3 as the dominant isoform of the IP₃ receptor in taste cells. IP₃ has been shown to be an important second messenger in both bitter and sweet taste transduction, and IP₃R3 likely mediates the Ca²⁺ release from intracellular stores in response to IP₃. In bitter taste transduction, many signaling components have been identified, and IP₃R3 is co-expressed in the same taste cells (Fig. 6). Bitter stimuli bind to T2R/TRB taste receptors coupled to a heterotrimeric G protein complex consisting of α-gustducin and its partners, β3 and γ13. Alpha gustducin activates PDE, causing decreases in intracellular cAMP, while its βγ partners stimulate PLCβ2 to produce IP₃ and DAG. IP₃ subsequently binds to IP₃R3, causing increases in cytosolic Ca²⁺, due to release from intracellular stores (Fig. 1B). The unique properties of IP₃R3, including its regulation by Ca²⁺ and cAMP dependent kinases, are consistent with known characteristics of bitter signaling in taste cells.