Immunocytochemical evidence for co-expression of Type III IP3 receptor with signaling components of bitter taste transduction

Abstract

Background: Taste receptor cells are responsible for transducing chemical stimuli into electrical signals that lead to the sense of taste. An important second messenger in taste transduction is IP3, which is involved in both bitter and sweet transduction pathways. Several components of the bitter transduction pathway have been identified, including the T2R/TRB taste receptors, phospholipase C beta2, and the G protein subunits alpha-gustducin, beta3, and gamma13. However, the identity of the IP3 receptor subtype in this pathway is not known. In the present study we used immunocytochemistry on rodent taste tissue to identify the IP3 receptors expressed in taste cells and to examine taste bud expression patterns for IP3R3.

Results: Antibodies against Type I, II, and III IP3 receptors were tested on sections of rat and mouse circumvallate papillae. Robust cytoplasmic labeling for the Type III IP3 receptor (IP3R3) was found in a large subset of taste cells in both species. In contrast, little or no immunoreactivity was seen with antibodies against the Type I or Type II IP3 receptors. To investigate the potential role of IP3R3 in bitter taste transduction, we used double-label immunocytochemistry to determine whether IP3R3 is expressed in the same subset of cells expressing other bitter signaling components. IP3R3 immunoreactive taste cells were also immunoreactive for PLCbeta2 and gamma13. Alpha-gustducin immunoreactivity was present in a subset of IP3R3, PLCbeta2, and gamma13 positive cells.

Conclusions: IP3R3 is the dominant form of the IP3 receptor expressed in taste cells and our data suggest it plays an important role in bitter taste transduction.

Figure 1

Diagrammatic representation of a rodent taste bud and important components of the bitter transduction pathway. (A) A typical taste bud consists of 50-100 taste receptor cells (TRCs) that extend from the basal lamina to the taste pore. Taste stimuli interact with taste receptors on the apical membrane, while nerve fibers form chemical synapses with the basolateral membrane. Basal cells (labeled B) along the margin of the taste bud are proliferative cells that give rise to taste receptor cells. (B) Bitter stimuli interact with T2R/TRB receptors located on the apical membrane. These receptors couple to a heterotrimeric G protein consisting of α-gustducin, β3, and γ13. Alpha gustducin activates phosphodiesterase (PDE), causing decreases in intracellular cAMP, while β3γ13 activates phospholipase C β2 (PLCβ2) to produce the second messengers inositol 1,4,5 trisphosphate (IP₃) and diacylglycerol (DAG). The IP₃ binds to receptors located on smooth endoplasmic reticulum, causing a release of Ca²⁺ into the cytosol. The purpose of this study was to identify the IP₃ receptor isotype that is expressed in taste cells.

Figure 2

Laser scanning confocal micrographs (LCSMs) of rat circumvallate taste buds labeled with antibodies against the three isoforms of the IP₃ receptor. (A). IP₃R1 immunoreactivity (IR), (B) IP₃R2 IR, (C) IP₃R3 IR, (D) no primary antibody control for IP₃R3. The scale bar in each figure represents 10 μm.

Figure 3

LCSMs of rat circumvallate taste buds double labeled with antibodies against α-gustducin (green) and IP₃R3 (red). Panel C shows an overlay of A and B with the differential interference contrast image to enhance visualization of individual taste cells. Scale bar is 10 μm. Note that all α-gustducin IR taste cells are also IP₃R3 IR, however not all IP₃R3 IR cells are α-gustducin IR.

Figure 4

LCSMs of rodent circumvallate taste buds double labeled with antibodies against PLCβ2 (green) and IP₃R3 (red). Panels A-C are from mouse; D-F are from rat. Panels C and F represent the overlay, as described for Figure 3. Scale bar is 20 μm. Note almost complete co-localization of IP₃R3 IR and PLCβ2 IR.

Figure 5

LCSMs of rodent circumvallate taste buds double labeled with antibodies against γ13 (green) and IP₃R3 (red). Panels A-C are from mouse; D-F are from rat. Panels C and F represent the overlay as described for Figs. 3-4. Scale bar is 20 μm. Note nearly complete co-localization of IP₃R3 IR and γ13 IR.

Figure 6

Qualitative representation of the co-expression patterns of IP₃R3, α-gustducin, PLCβ2, and γ13 in circumvallate taste buds.