| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1Department of Biological Sciences, University at Buffalo, The State University of New York, Buffalo, New York; and 2Rocky Mountain Taste and Smell Center Neuroscience Program and Department of Cell and Developmental Biology, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado
Submitted 10 August 2007; accepted in final form 11 January 2008
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
; Rossler et al. 1998). Disruption of this signaling pathway impairs the ability to detect multiple taste stimuli in mice (Zhang et al. 2003).
Recent studies suggest that two functional classes exist in taste cells (Clapp et al. 2006; DeFazio et al. 2006; Medler et al. 2003). Some taste cells express synaptic terminal associated proteins such as voltage-gated calcium channels (VGCCs) (Medler et al. 2003) and SNAP-25 (Yang et al. 2000). Other cells express signaling molecules that include taste GPCRs, PLCβ2, and IP3 receptors (IP3R3) (Clapp et al. 2001, 2006; DeFazio et al. 2006). Taste cells expressing the PLCβ2/IP3R3 signaling pathway do not express proteins necessary to form conventional synapses (Clapp et al. 2004, 2006; DeFazio et al. 2006) but instead release neurotransmitters through hemichannels, likely pannexins (Huang et al. 2007; Romanov et al. 2007).
Conventional synapses are present in some taste cells, although it is not clear which taste qualities these cells transduce. Since salty and sour taste qualities were intact in the PLCβ2 knock-out mouse (Zhang et al. 2003), it is possible that taste cells with conventional synapses detect these taste qualities. This is supported by the report that acid evoked responses are generated by calcium influx through VGCCs (Richter et al. 2003). Taken together, these data suggest that taste receptor cells can be grouped into two separate populations: 1) cells that depend on calcium influx through VGCCs to communicate via conventional synapses and 2) cells that use GPCR-dependent second messenger pathways to transduce signals but lack conventional synapses.
Since growing evidence supports the presence of two distinct signaling mechanisms in taste cells, the initial purpose of this study was to characterize the evoked calcium responses in taste cells when stimulated by bitter compounds and cell depolarization. Increasing extracellular K+ caused cell depolarization and subsequent calcium influx through VGCCs, whereas bitter stimuli activated PLC with concomitant calcium release from intracellular stores. Using an IP3R3-green fluorescent protein (GFP) mouse to identify taste cells without conventional synapses, we found significant differences in the calcium responses to these different stimuli. Most responsive taste cells were sensitive to either high KCl or bitter. Quite unexpectedly, we found a third group of taste cells that were responsive to both high KCl and bitter stimuli. These taste cells did not express IP3R3, indicating that they respond to bitter stimuli through a PLCβ2/IP3R3-independent pathway. Analysis of these taste cells determined that the bitter responses were inhibited by U73122 [GenBank] , suggesting another PLC isoform mediates these responses. We found that PLCβ3 and IP3R1 are also expressed in taste cells, suggesting that multiple PLC signaling pathways are used to transduce bitter stimuli in mice.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
All measurements of intracellular Ca2+ were performed in isolated taste cells. Taste receptor cells were harvested from the circumvallate and foliate papillae of adult C57BL/6 mice and FVB transgenic mice that express GFP driven by the IP3R3 promoter. Animals were cared for in compliance with the University at Buffalo Animal Care and Use Committee. Mouse taste receptor cells were isolated from lingual epithelium as previously described (Medler et al. 2003). All chemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise noted.
Generation of the IP3R3-tauGFP transgenic mouse
The IP3R3-tauGFP transgenic mouse construct was made by linking a 3.7-kb IP3R3 proximal promoter (Tamura et al. 2001) to a cassette encoding for tauGFP (amplified from the IRES-tauGFP-LNL plasmid from the Mombaerts laboratory) (Rodriguez et al. 1999). The IP3R3 promoter (–3.7 kb to –16 bp relative to transcription start site) was amplified by PCR using a BAC clone as template (BAC 27777 obtained from Incyte Genomics). The Kozak consensus sequence of tau was modified (A 
C at –4 from start) using PCR to improve the translational efficiency of tauGFP (Kozak 1994, 2002). Further modification of the tauGFP cassette included the addition of three polyadenylation sites (Maxwell et al. 1989). The vector was cloned into pBluescript II SK(+) (Stratagene, La Jolla, CA), and the construct was verified by sequencing. The IP3R3-tauGFP transgenic mouse construct was microinjected into fertilized oocytes by personnel in the University of Colorado Health Sciences Center transgenic facility following standard procedures (Hogan et al. 1986). We obtained five positively genotyped founder pups and one of these transgenic lines expressed tauGFP in select cells within the taste bud.
Immunocytochemistry of IP3R3-GFP mice
Transgenic mice (IP3R3-GFP) were perfused with 4% paraformaldehyde/0.1 M phosphate buffer (PB; Electron Microscopy Sciences, Ft. Washington, PA). Following perfusion, tongues were removed and placed into 4% paraformaldehyde/0.1 M PB for 1 h followed by a 4°C overnight incubation in 20% sucrose (in 0.1 M PB). For some experiments, tongues were immersion fixed overnight in 4% paraformaldehyde/0.1 M PB and cryoprotected in 20% sucrose/0.1 M PB. Forty micrometer sections were cut and washed in PBS three times for 10 min each at room temperature. Antigen retrieval was performed by placing sections in 10 mM sodium citrate at 80°C for 15 min. This was done to help disrupt protein cross-bridges formed by formalin fixation and expose antigen binding sites (Evers et al. 1998). All sections were incubated in blocking solution (0.3% Triton X-100, 1% normal goat serum, and 1% bovine serum albumin in 0.1 M PBS) for 1–2 h at room temperature. Sections from mouse circumvallate papillae were incubated with primary antibodies overnight at 4°C. Controls with no primary antibodies were included in each experiment.
Mouse anti-IP3R3 (Transduction Labs, Lexington, KY) was used at 1:50 in blocking solution following antigen retrieval, whereas rabbit anti-IP3R1 (Affinity Bioreagents, Golden, CO) was used at 1:200 in blocking solution. Rabbit anti-PLCβ2 (Santa Cruz Laboratories, Santa Cruz, CA) was used at 1:1,000 in blocking solution, rabbit anti-PLCβ3 (Santa Cruz Laboratories) was diluted 1:400, mouse anti-SNAP 25 (Genway Biotech, San Diego, CA) was used at 1:200 in blocking solution, and PGP9.5 (Biogenesis, Kingston, NH) was diluted to 1:250 in blocking solution. Following overnight incubation in primary antibodies, sections were washed three times for 10 min in PBS. Sections were incubated with the appropriate secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature in the dark for 2 h. Following incubation with secondary antibodies, sections were washed again, mounted on RITE-ON micro slides (Becton Dickinson, Portsmouth, NH) using Flouromount-G (Southern Biotechnology Associates, Birmingham, AL), and coverslipped.
Sections were viewed with a three-channel laser scanning confocal with Krypton-Argon lasers on a Nikon Diaphot 200. Sequential scanning techniques were used. Images were captured with a cooled CCD camera, and Axiovision software was used for data acquisition. Images were processed using Adobe Photoshop CS software adjusting only brightness and contrast.
Behavioral procedures
To determine whether GFP expression affected the taste preferences of the transgenic mice compared to wild-type FVB mice or C57BL/6 mice, mice from each group were subjected to two bottle preference tests using the protocol described in Ruiz et al. (2003), except that no Latin Square model was used to determine stimulus presentation. Each test concentration was presented in conjunction with water for a total of 48 h. Test solutions were switched with water every 24 h to ensure no side preferences developed. Preference ratios were calculated as volume of test solution intake/total volume intake (test solution + water). Five male mice from each background (C57BL/6, FVB wild-type, FVB IP3R3-GFP) were used for a total of 15 mice. Three taste stimuli were tested, sucrose, sodium chloride (NaCl), and denatonium, and each stimulus was presented at four concentrations in ascending order. The concentrations used were as follows: 1) denatonium 0.01, 0.1, 1, and 10 mM; 2) sucrose 0.5, 5, 50, and 150 mM; and 3) NaCl 1, 10, 100, and 300 mM. Preference ratios for each mouse strain were compared using repeated measures two-way ANOVA. Further analysis was performed using simple effects and Student's t-test to determine significant differences. A value of P < 0.05 was determined as the limit of significance.
Calcium imaging
All measurements of intracellular Ca2+ were performed in isolated taste cells. Isolated taste receptor cells were loaded for 40 min with 2 µM fura 2-AM (Molecular Probes, Invitrogen, Carlsbad, CA) containing the nonionic dispersing agent Pluronic F-127 (Molecular Probes, Invitrogen). Loaded cells were visualized using an Olympus Ix71 microscope with a 40x oil immersion lens, and images were captured using a Sensicam QE camera (Cooke Corp, Romulus, MI). Excitation wavelengths of 340 and 380 nm were used with an emission wavelength of 510 nm. During experiments, cells were kept under constant perfusion, and images were collected every 4 s using Imaging Workbench 5.2 (Indec Biosystems, Santa Clara, CA). Faster capture rates resulted in damage to the cells. Since our shuttering rate was relatively slow, a faster sampling (0.5 s) was done on a subset of cells to determine whether we were accurately measuring the peak fluorescence increases (data not shown). There was no difference in the peak amplitude of the response in taste cells when images were captured every 4 s compared with the images captured every 0.5 s, indicating that peak fluorescence responses could accurately be measured at the slower sampling rate. Experiments were graphed and analyzed using OriginPro 7.5 software.
Calcium levels were collected as a ratio of fluorescence intensities. Fluorescence values were calibrated using the Fura-2 Calcium Imaging Calibration kit (Invitrogen). The effective dissociation constant, Kd was calculated to be 253 nM, which was used in the following equation to calculate calcium concentration
 |
 |
Control experiments were performed to ensure that the GFP expression was not producing a confounding signal that interfered with the fura2 measurements. The GFP construct is comprised of EGFP1, which has a peak excitation wavelength close to 488 nm and should not be greatly excited at 340 or 380 nm. For all experiments, filters with a narrow band-pass were used to reduce nonspecific excitation. As a result, GFP fluorescence was not detectable using the fura2 filters in control runs that excited GFP-expressing cells which were not loaded with fura2-AM. In these experiments, only background fluorescence was detected.
Responses to a high potassium solution (Tyrode's with 50 mM NaCl replaced by 50 mM KCl) and a bitter mixture consisting of 10 mM denatonium benzoate and 500 µM cycloheximide were initially characterized. We performed a concentration dilution series for both denatonium and cycloheximide and determined that 10 mM denatonium benzoate and 0.5 mM cycloheximide gave a saturating response. Lower concentrations of the bitter mixture generated smaller calcium responses in the same cells (see Supplemental Fig. S4),1 whereas higher concentrations evoked comparable responses.
All solutions were bath applied using a gravity flow perfusion system (Automate Scientific, San Francisco, CA) and laminar flow perfusion chambers (RC-25F, Warner Scientific, Hamden, CT). An evoked response was defined as measurable if the increase in fluorescence was >2 SD above baseline. The duration of the response to 50 mM KCl was measured at half-maximal response to preclude measuring the plateau phase. Since there was not a distinct plateau phase in the bitter-evoked responses, the entire duration of those responses was included in the measurements. Statistical comparisons were made using an unpaired Student's t-test with a limit of significance at P < 0.05. Pearson's 
2 analysis was performed to detect any significant differences between the numbers of responsive taste cells across mouse strains and papillae types tested.
RT-PCR analysis of isolated taste tissue
Isolated taste buds from the circumvallate and foliate papillae were lysed, and RNA was purified using the RNeasy Mini Kit from Qiagen (Valencia, CA) according to the manufacturer's instructions. Total RNA isolated from brain tissue was used as a positive control. PCR analysis for GAPDH was performed in a 50-µl reaction using 3 µl cDNA from each sample to determine sample quality and to check for genomic contamination. If any genomic DNA was present, the sample was discarded and a new sample was collected. cDNA from isolated taste buds was tested for the expression of PLC isoforms. Previously published primers to conserved regions within the X and Y catalytic domains of PLCs (Lee et al. 1993) were used to check for the expression of PLC isoforms in taste cells. The identity of all PCR products was confirmed by DNA sequencing.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
The transgenic IP3R3-GFP mice used in this study were generated in an FVB background, whereas the majority of the previous studies characterizing the PLCβ2 pathway in taste cells have been performed in C57BL/6 mice. Since mouse strain differences can affect taste perception (Bachmanov et al. 1996, 1998a,b; Manita et al. 2006; Ninomiya et al. 1984a,b) and could potentially affect taste cell structure and/or function, immunocytochemical and behavioral analyses were performed to determine if the IP3R3-GFP mice were comparable to C57BL/6 mice.
Control experiments were performed to determine how well GFP expression correlated with immunoreactivity for the IP3R3 protein. Figure 1A shows sections obtained from the circumvallate papillae of an IP3R3-GFP mouse labeled with an antibody raised against IP3R3. Analysis of 40 taste buds from four GFP-positive mice (cells, 178) found that 94% of GFP labeled cells were IP3R3-immunoreactive (-IR), whereas 97% of IP3R3-IR cells expressed GFP.
|
, 2004; Miyoshi et al. 2001). Immunocytochemical analysis of PLCβ2 expression in IP3R3-GFP cells (Fig. 1B) found that 83% PLCβ2-IR cells expressed GFP, whereas 90% of the GFP cells had PLCβ2 immunoreactivity (46 taste buds, 4 mice, 185 cells). This level of overlap between IP3R3-GFP and PLCβ2-IR is comparable to the co-localization of IP3R3 and PLCβ2 in the circumvallate papillae of a C57BL/6 mouse (Fig. 1C). Although co-localization of GFP and IP3R3-IR or PLCβ2-IR was not 100% in the transgenic mouse, the GFP-expressing cells are representative of the taste cells that use the PLCβ2/IP3R3 signaling pathway.
IP3R3-IR taste cells lack chemical synapses (Clapp et al. 2004) and SNAP25 expression correlates with chemical synapses in circumvallate taste cells (Yang et al. 2000), which indicate that IP3R3 and SNAP25 are expressed in separate taste cell populations; however, DeFazio et al. (2006) detected mRNA transcripts for both IP3R3 and SNAP25 in some taste cells, but did not report on protein expression. Therefore we labeled IP3R3-GFP mice with anti-SNAP25 antibodies to determine whether any overlap existed between IP3R3-GFP–and SNAP25-expressing taste cells. We found no co-localization between SNAP-25 expression and IP3R3-GFP (Fig. 2A). The circumvallate taste cells from the IP3R3-GFP mice were also labeled with anti-PGP9.5 to assess the potential co-localization of these two proteins. In rats, PGP9.5 labels taste cells that do not express components of the PLC signaling pathway (Yee et al. 2001) and does not co-localize with IP3R3 (Clapp et al. 2004). Therefore we would expect PGP9.5 and IP3R3-GFP to be localized in different taste cells. These experiments detected no overlap between PGP9.5 expression and IP3R3-GFP taste cells (Fig. 2B).
|
, 2004, 2006; DeFazio et al. 2006; Medler et al. 2003; Miyoshi et al. 2001; Yee et al. 2001). Characterization of evoked responses
Studies have reported that taste stimuli can cause either calcium influx through VGCCs or calcium release from intracellular stores in taste cells (Clapp et al. 2006; DeFazio et al. 2006; Richter et al. 2003; Zhang et al. 2003). However, any differences in the types of calcium responses that these stimuli provoke have not been determined. Our initial studies were performed to describe the evoked calcium responses that originate from either calcium release from internal stores or high K+ induced calcium influx. Experiments compared the responsiveness of taste cells to high K+ that depolarizes excitable cells and causes VGCCs to open (Wei and Chiang 1986) and to bitter stimuli that activates a PLC signaling pathway and causes calcium release from internal stores (Akabas et al. 1988; Huang et al. 1999; Ogura et al. 2002; Yan et al. 2001). We know that applying 50 mM KCl results in opening of VGCCs in taste cells, because this response is inhibited by 200 µM CdCl2 (Supplemental Fig. S2), a known blocker of VGCCs (Beam and Knudson 1988). Most experiments characterizing the types of calcium responses generated by these two stimuli were performed on the IP3R3-GFP transgenic mouse line, but C57BL/6 mice were also used in some experiments. Because the purpose of experiments in this section is to compare the overall responsiveness of all taste cells, responses from IP3R3-GFP mice include both GFP-expressing taste receptor cells and non–GFP-expressing taste receptor cells. If an experiment was exclusively reporting results from GFP expressing taste cells, it is noted in the text and the figure legend.
A mixture of bitter compounds (10 mM denatonium, 500 µM cycloheximide) was applied to isolated taste receptor cells to activate bitter GPCRs and the PLC signaling pathway, whereas 50 mM KCl was used to activate calcium influx through VGCCs. Taste cells were loaded with the calcium-sensitive fluorescent dye fura2-AM (Fig. 3C), and stimulus-evoked calcium changes in isolated taste receptor cells were measured. In Fig. 3, two different isolated taste cells were subjected to an application of the bitter mixture (denoted by the black bar) followed by 50 mM KCl (denoted by the arrow). In Fig. 3A, the taste cell did not respond to the bitter stimulus, but 50 mM KCl caused a large increase in cytosolic calcium. In Fig. 3B, the taste cell did not respond to 50 mM KCl, but the bitter stimuli caused an increase in intracellular calcium. While many cells did not respond to either stimulus, the responses shown in Fig. 3, A and B, are representative of the responses obtained in this study.
|
Although there were notable differences between the types of calcium responses these stimuli evoked, other comparisons did not reveal any significant differences. Different mouse strains can exhibit variable sensitivities to taste qualities (Bachmanov et al. 1996, 1998a,b; Manita et al. 2006; Ninomiya et al. 1984a,b), but we did not detect any differences in the evoked responses (bitter or 50 mM KCl) between C57BL/6 and IP3R3-GFP mice (P = 0.83). Since multiple cranial nerves innervate taste papillae and have different response profiles to taste stimuli (Danilova and Hellekant 2003), we also compared the evoked responses from circumvallate and foliate papillae. We found no significant differences for bitter evoked responses (P = 0.065) or 50 mM KCl–evoked responses (P = 0.15). We also determined if there were differences in the percentage of taste cells that responded to each stimulus as a function of mouse strain or papillae type. These data are summarized in Supplemental Fig. S3. 2 analysis of the number of bitter responses found no significant differences between the overall percentage of bitter responsive cells in C57Bl/6 mice compared with IP3R3-GFP mice (P = 0.24) and no significant differences between the overall percentage of 50 mM KCl–responsive taste cells between IP3R3-GFP mice and C57Bl/6 mice (P = 0.35). Based on these data, we concluded that 50 mM KCl and bitter stimuli evoke significantly different calcium responses but that mouse strain and papillae type do not impact the type of calcium response generated.
IP3R3-GFP–positive taste cells respond to bitter stimuli but not to depolarization with high K+
Based on published studies (Clapp et al. 2004, 2006; DeFazio et al. 2006), we predicted that at least some of the IP3R3-GFP positive cells would be sensitive to bitter stimulation but that 50 mM KCl would not elicit a response. As expected, 50 mM KCl failed to elicit a calcium response in all IP3R3-GFP cells tested (n = 56). However, 79% of the IP3R3-GFP–expressing taste cells tested (n = 24) were bitter responsive. This relative high percentage of responsive taste cells was not surprising since we preselected for taste cells that express the PLCβ2/IP3R3 signaling pathway and were therefore likely to be responsive to complex stimuli. Since most of the tested IP3R3-GFP–positive taste cells (n = 19 of 24) were isolated from the circumvallate papillae, which have been shown to be more bitter sensitive than other papillae (Danilova and Hellekant 2003), it is not surprising that many of these taste cells were bitter sensitive. Figure 4 shows the bitter-induced calcium elevation and the corresponding lack of response to 50 mM KCl in an IP3R3-GFP expressing taste cell.
|
Quite unexpectedly, we found that some isolated taste cells responded to both bitter stimuli and 50 mM KCl (Fig. 5A). This finding is surprising because earlier studies (Clapp et al. 2004, 2006; DeFazio et al. 2006; Zhang et al. 2003) have concluded that bitter responsive taste cells are entirely separate from taste cells that express VGCCs. We designated these taste cells as "dual responsive," and pair-wise Student's t-test determined that the bitter evoked responses were significantly different from the 50 mM KCl responses (mean bitter response = 326 nM, mean 50 mM KCl response = 2,662 nM; P = 0.0095) in these cells. These significant differences were comparable to the results from taste cells that respond to a single stimulus. We found that 68% of taste with VGCCs were responsive to bitter stimuli (n = 48 of 70), whereas 29% of bitter responsive cells (n = 48 of 167) were sensitive to 50 mM KCl application. These dual responsive cells were present in both the IP3R3-GFP transgenic and C57BL/6 mice. Importantly, all responses were measured in isolated taste receptor cells, ruling out a role for gap junctions and cell to cell communication through neurotransmitter release in our study. In the IP3R3-GFP mice, all of the dual responsive cells were GFP negative, indicating that they did not express the PLCβ2/IP3R3 signaling pathway.
|
Figure 5B reports the percentage of dual responsive taste cells compared with the total number of responsive cells for a particular stimulus. The total number of bitter responsive cells was normalized to 100%, whereas the number of bitter responsive cells that also responded to 50 mM KCl was normalized to the same value. Of the tested cells, 29% of the bitter responsive cells were dual responsive. However, in the taste cells that responded to 50 mM KCl, 69% of the 50 mM KCl–responsive cells also responded to bitter stimuli. The Venn diagram in Fig. 5C summarizes the percentage of dual responsive taste cells for each stimulus type.
Dual responsive cells respond to bitter stimuli through a PLC signaling pathway
Our data from the IP3R3-GFP mice indicate that PLCβ2 is not expressed in taste cells that express VGCCs, which agrees with other studies (Clapp et al. 2004, 2006; DeFazio et al. 2006). Therefore the dual responsive cells must be using a PLCβ2/IP3R3-independent pathway to detect bitter tastants. Analysis of the bitter response in dual responsive taste cells found that this response is inhibited by the PLC inhibitor, U73122
[GenBank]
(1 µM, n = 5; Fig. 6A), indicating the presence of another PLC isoform. Further analyses using a paired Student's t-test determined that this bitter response was not significantly affected by U73433
[GenBank]
(average = 164 nM before U73433
[GenBank]
, average = 170 nM after U73433
[GenBank]
, P = 0.868, n = 3), whereas U73122
[GenBank]
inhibited the response. The amplitude of the 50 mM KCl-dependent response in these dual responsive taste cells was not significantly affected by U73433
[GenBank]
(average = 344 nM before U73433
[GenBank]
, average = 264 nM after U73433
[GenBank]
, P = 0.619, n = 3) or U73122
[GenBank]
(average = 650 nM before U73122
[GenBank]
, average = 463 nM after U73122
[GenBank]
, P = 0.291, n = 3). Addition of thapsigargin, which prevents the refilling of internal calcium stores, eliminated the bitter evoked calcium response (2 µM; Fig. 6B) but did not significantly impact the 50 mM KCl response (average = 600 nM before thapsigargin, average = 386 nM after thapsigargin, P = 0.181, n = 3), indicating that the bitter evoked response depends on calcium release from internal stores, whereas the high KCl response does not. To confirm this result, stimuli were applied in calcium-free external solution. We found that bitter-induced responses were still present in calcium-free external solution, but hi K–induced responses were abolished (Fig. 6C; n = 7). Therefore in dual responsive taste cells, bitter evoked calcium changes depend on calcium release from internal calcium stores likely through a PLC-mediated pathway, whereas 50 mM KCl-dependent responses rely on calcium influx across the plasma membrane.
|
Since immunocytochemical data indicate that PLCβ2 is primarily restricted to IP3R3-GFP taste cells, our data indicated that additional PLC isoforms are expressed in the dual responsive taste receptor cells. Therefore mRNA isolated from C57BL/6 mice taste cells was analyzed to determine whether multiple PLC isoforms are present. A degenerate PCR primer pair taken from Lee et al. (1993) amplified two PCR products in taste cells that were harvested from circumvallate (CV) and foliate (Fol) papillae (Fig. 7). Sequence analysis of these two PCR products determined that the smaller 850-bp PCR product shared 99% identity with PLCβ2, whereas the larger 1-kb PCR product was identified as PLCβ3 with 99% identity. Immunocytochemical analysis of taste buds in the circumvallate papillae of an IP3R3-GFP mouse (Fig. 8A) and a C57BL/6 mouse (Fig. 8C) found robust anti-PLCβ3 labeling. No discernable overlap was found between PLCβ3-IR and GFP expression in Fig. 8A, signifying that PLCβ3 and IP3R3 are present in separate populations of taste cells. Further immunocytochemical analysis detected IP3R1-IR in mouse taste receptor cells (Fig. 8, B and D) that was largely nonoverlapping with IP3R3-GFP (Fig. 8B). Negative control sections shown on the right of each panel did not have any immunoreactivity. Dual labeling experiments with anti-PLCβ3 and anti-IP3R1 determined that the expression of these proteins heavily overlaps with each other but much less so with IP3R3-GFP (Fig. 9). Immunocytochemical analysis with anti-SNAP 25, anti-PLCβ3, and anti-IP3R1 found some overlap in the expression of these proteins (data not shown). Although not completely overlapping, there were many PLCβ3-IR and IP3R1-IR taste cells that also were immunoreactive for SNAP-25, signifying that PLCβ3 and IP3R1 can be found in taste cells with conventional synapses.
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
; Behe et al. 1990; Bernhardt et al. 1996; DeFazio et al. 2006; Maruyama et al. 2006; Ogura et al. 2002; Richter et al. 2003), there has been little work describing the differences between the types of calcium responses evoked by these stimuli. Bitter, sweet, and umami stimuli cause an increase in [Ca2+]i that is dependent on activation of the PLCβ2/IP3R3 signaling pathway (Zhang et al. 2003). These taste cells do not express the proteins associated with chemical synapses (Clapp et al. 2004, 2006; DeFazio et al. 2006) but instead depend on a unique mechanism of neurotransmitter release through the activation of a hemichannel (Huang et al. 2007; Romanov et al. 2007). However, other taste cells have conventional synapses and depend on calcium influx through VGCC to generate signals (Behe et al. 1990; DeFazio et al. 2006; Medler et al. 2003; Richter et al. 2003). To date, possible differences between these stimulus-evoked calcium responses have not been characterized but presumably significantly different calcium responses may be needed to activate these distinct neurotransmitter release mechanisms. Identifying potential differences in the evoked calcium responses will increase our understanding of how taste cells produce distinct stimulus signals that are transmitted to the brain.
We initially characterized the types of evoked cytosolic calcium changes in taste cells in response to either bitter stimuli or VGCC activation and found that these two response profiles are significantly different from each other. Opening of VGCCs caused a significantly faster and larger calcium elevation compared with bitter evoked responses. This result was not surprising since these stimuli activate signaling pathways that access two different calcium sources to generate a response. We expected the calcium influx through VGCC to cause a large, fast response since the driving force for calcium to enter the cell is very large and occurs as soon as VGCCs open (Hille 2001), whereas activating a GPCR/second messenger pathway causes calcium release from stores and a smaller, slower change in cytosolic calcium (see Fig. 3). Further comparisons determined that mouse strain and papillae type did not influence the type of calcium response evoked by each stimulus, indicating that the type of calcium response generated in a taste cell is caused solely by the activation of a particular signaling pathway. The ability to produce unique signals in response to different stimuli provides taste cells with the capacity to convey complex signals to afferent gustatory neurons. The complexity of these signals may have important implications in taste coding.
One surprising finding in our study was the relatively large number of bitter responses that we recorded compared with reported findings from other studies (Caicedo and Roper 2001; Caicedo et al. 2002, 2003). Since we were trying to characterize the bitter evoked response, we used higher concentrations of bitter compounds to ensure that we were detecting maximal responses in bitter sensitive cells. Dose–response curves of different concentrations of the bitter compounds found that less concentrated bitter mixtures generated smaller changes in cytosolic calcium that are more difficult to detect (see Supplemental Fig. S4). Therefore we may have detected bitter sensitive taste cells that were missed in previous studies. We believe that the responses to the bitter mixture were specific because many taste receptor cells were tested that did not respond (Figs. 3 and S3) and the bitter evoked calcium elevations were selectively inhibited by pharmacological treatment (Fig. 6).
Overwhelming evidence has shown that the transduction of stimuli through the PLCβ2/IP3R3 signaling pathway occurs in taste cells that do not express the proteins associated with chemical synapses (Clapp et al. 2004, 2006; DeFazio et al. 2006; Tomchik et al. 2007), which is further supported by our finding that these taste cells do not express VGCCs. Instead, PLCβ2/IP3R3-expressing taste cells use a distinct signaling pathway that depends on the calcium-dependent opening of a hemichannel for neurotransmitter release (Huang et al. 2007; Romanov et al. 2007). This differs from acid sensitive cells that have been shown to depend on calcium influx through VGCC to transmit signals (Richter et al. 2003), presumably through conventional synapses. These data show that there are at least two functional classes of taste cells that use distinct signaling mechanisms to transmit signals to the brain. It has recently been proposed that taste cells expressing the PLCβ2/IP3R3 signaling pathway may be communicating with taste cells expressing VGCCs. This hypothesis was based on the finding in taste cell slice preparations that PLCβ2/IP3R3-expressing cells are selective in their responsiveness to different tastants but that taste cells expressing VGCCs are more broadly tuned and respond to tastants that activate the PLCβ2 signaling pathway (Tomchik et al. 2007).
However, our data indicate the existence of a third functional class of taste cells. Approximately 28% of the bitter responsive taste cells were also sensitive to depolarization by 50 mM KCl. In contrast, 66% of taste cells that responded to high KCl were bitter responsive (Fig. 5). All of these taste receptor cells were isolated and were not in contact with other taste receptor cells. Since it has been shown that PLCβ2 is not expressed in taste cells that have voltage-dependent calcium influx (Clapp et al. 2004; DeFazio et al. 2006; Tomchik et al. 2007), these data indicate that there is a PLCβ2/IP3R3-independent pathway detecting bitter stimuli in taste cells with VGCCs. Analysis of the evoked responses in these dual responsive cells determined that the bitter and 50 mM KCl responses were significantly different from each other but were comparable to the evoked bitter and 50 mM KCl responses found in other taste cells. These data show that the evoked calcium response to a particular stimulus is specifically generated by the cell to convey unique signals to the brain. Furthermore, a single taste receptor cell can generate significantly different responses depending on the initial stimulus signal.
Specific characterization of the bitter evoked response in the dual responsive cells found that it was inhibited by U73122 [GenBank] (Fig. 6A) and thapsigargin (Fig. 6B), signifying these responses are dependent on PLC activation and calcium release from internal stores. These bitter responses were still present in the absence of external calcium, but 50 mM KCl responses were inhibited under these conditions (Fig. 6C), which confirms that the bitter evoked cytosolic calcium increases depend on calcium release from internal stores, whereas the 50 mM KCl–evoked response depends on external calcium. Therefore unlike other taste receptor cells, the dual responsive taste cells can use both calcium release from internal stores and calcium influx across the plasma membrane to produce stimulus-evoked calcium signals.
Since the physiological analysis of these dual responsive taste cells indicated the involvement of another PLC isoform in the transduction of bitter stimuli, we used RT-PCR analysis of mRNA from groups of isolated taste buds to detect the potential expression of other PLC isoforms in taste cells. We identified PLCβ2 and PLCβ3 (Fig. 7) in these cells, both of whom associate with GPCR-dependent signaling pathways and could potentially be involved in the transduction of taste stimuli. Staining of circumvallate papillae with anti-PLCβ3 revealed labeling in a distinct population of taste receptor cells (Figs. 8, A and C, and 9).
Although PLCβ2 is clearly essential for the normal transduction of multiple taste qualities (Zhang et al. 2003), we hypothesize that a PLCβ3 signaling pathway is present in the dual responsive taste receptor cells and also contributes to the detection of taste stimuli. IP3R1 expression is present in mouse taste cells (Fig. 8, B and D) and co-expresses with PLCβ3 (Fig. 9), further supporting the presence of an additional PLC signaling pathway. This differs with what has been reported in rat taste cells (Clapp et al. 2001; Miyoshi et al. 2001), and although Miyoshi et al. (2001) did detect IP3R1 transcripts through RT-PCR, their in situ analysis of the rat taste cells did not reveal any significant labeling. We conclude that mouse taste receptor cells can use multiple signaling pathways to transduce bitter signals. Although both of these signaling pathways depend on PLC activation and calcium release from stores, they seem to use distinct downstream signaling mechanisms to communicate signals to the brain. Further study is needed to determine if this is true for other stimuli.
|
GRANTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
1 The online version of this article contains supplemental data. 
Address for reprint requests and other correspondence: K. Medler, Dept. of Biological Sciences, Univ. at Buffalo, The State University of New York, Buffalo, NY 14260 (E-mail: kmedler{at}buffalo.edu)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
Bachmanov AA, Schlager G, Tordoff MG, Beauchamp GK. Consumption of electrolytes and quinine by mouse strains with different blood pressures. Physiol Behav 64: 323–330, 1998a.[CrossRef][Medline]
Bachmanov AA, Tordoff MG, Beauchamp GK. Ethanol consumption and taste preferences in C57BL/6ByJ and 129/J mice. Alcohol Clin Exp Res 20: 201–206, 1996.[CrossRef][Web of Science][Medline]
Bachmanov AA, Tordoff MG, Beauchamp GK. Voluntary sodium chloride consumption by mice: differences among five inbred strains. Behav Genet 28: 117–124, 1998b.[CrossRef][Web of Science][Medline]
Beam KG, Knudson CM. Calcium currents in embryonic and neonatal mammalian skeletal muscle. J Gen Physiol 91: 781–798, 1988.
Behe P, DeSimone JA, Avenet P, Lindemann B. Membrane currents in taste cells of the rat fungiform papilla. Evidence for two types of Ca currents and inhibition of K currents by saccharin. J Gen Physiol 96: 1061–1084, 1990.
Bernhardt SJ, Naim M, Zehavi U, Lindemann B. Changes in IP3 and cytosolic Ca2+ in response to sugars and non-sugar sweeteners in transduction of sweet taste in the rat. J Physiol 490: 325–336, 1996.
Caicedo A, Kim KN, Roper SD. Individual mouse taste cells respond to multiple chemical stimuli. J Physiol 544: 501–509, 2002.
Caicedo A, Pereira E, Margolskee RF, Roper SD. Role of the G-protein subunit alpha-gustducin in taste cell responses to bitter stimuli. J Neurosci 23: 9947–9952, 2003.
Caicedo A, Roper SD. Taste receptor cells that discriminate between bitter stimuli. Science 291: 1557–1560, 2001.
Clapp TR, Medler KF, Damak S, Margolskee RF, Kinnamon SC. Mouse taste cells with G protein-coupled taste receptors lack voltage-gated calcium channels and SNAP-25. BMC Biol 4: 7, 2006.[CrossRef][Medline]
Clapp TR, Stone LM, Margolskee RF, Kinnamon SC. Immunocytochemical evidence for co-expression of Type III IP3 receptor with signaling components of bitter taste transduction. BMC Neurosci 2: 6, 2001.[CrossRef][Medline]
Clapp TR, Yang R, Stoick CL, Kinnamon SC, Kinnamon JC. Morphologic characterization of rat taste receptor cells that express components of the phospholipase C signaling pathway. J Comp Neurol 468: 311–321, 2004.[CrossRef][Web of Science][Medline]
Danilova V, Hellekant G. Comparison of the responses of the chorda tympani and glossopharyngeal nerves to taste stimuli in C57BL/6J mice. BMC Neurosci 4: 5, 2003.[CrossRef][Medline]
DeFazio RA, Dvoryanchikov G, Maruyama Y, Kim JW, Pereira E, Roper SD, Chaudhari N. Separate populations of receptor cells and presynaptic cells in mouse taste buds. J Neurosci 26: 3971–3980, 2006.
Dotson CD, Roper SD, Spector AC. PLCbeta2-independent behavioral avoidance of prototypical bitter-tasting ligands. Chem Senses 30: 593–600, 2005.
Evers P, Uylings HB, Suurmeijer AJ. Antigen retrieval in formaldehyde-fixed human brain tissue. Methods 15: 133–140, 1998.[CrossRef][Web of Science][Medline]
Hille B. Ion Channels of Excitable Membranes. Sunderland, MA: Sinauer Associates, 2001.
Hogan B, Constantini F, Lacy, E. Manipulating the Mouse Embryo. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1986.
Huang L, Shanker YG, Dubauskaite J, Zheng JZ, Yan W, Rosenzweig S, Spielman AI, Max M, Margolskee RF. Ggamma13 colocalizes with gustducin in taste receptor cells and mediates IP3 responses to bitter denatonium. Nat Neurosci 2: 1055–1062, 1999.[CrossRef][Web of Science][Medline]
Huang YJ, Maruyama Y, Dvoryanchikov G, Pereira E, Chaudhari N, Roper SD. The role of pannexin 1 hemichannels in ATP release and cell-cell communication in mouse taste buds. Proc Natl Acad Sci USA 104: 6436–6441, 2007.
Kozak M. Features in the 5' non-coding sequences of rabbit alpha and beta-globin mRNAs that affect translational efficiency. J Mol Biol 235: 95–110, 1994.[CrossRef][Web of Science][Medline]
Kozak M. Pushing the limits of the scanning mechanism for initiation of translation. Gene 299: 1–34, 2002.[CrossRef][Web of Science][Medline]
Lee CW, Park DJ, Lee KH, Kim CG, Rhee SG. Purification, molecular cloning, and sequencing of phospholipase C-beta 4. J Biol Chem 268: 21318–21327, 1993.
Manita S, Bachmanov AA, Li X, Beauchamp GK, Inoue M. Is glycine "sweet" to mice? Mouse strain differences in perception of glycine taste. Chem Senses 31: 785–793, 2006.
Maruyama Y, Pereira E, Margolskee RF, Chaudhari N, Roper SD. Umami responses in mouse taste cells indicate more than one receptor. J Neurosci 26: 2227–2234, 2006.
Maxwell IH, Harrison GS, Wood WM, Maxwell F. A DNA cassette containing a trimerized SV40 polyadenylation signal which efficiently blocks spurious plasmid-initiated transcription. Biotechniques 7: 276–280, 1989.[Web of Science][Medline]
Medler KF, Margolskee RF, Kinnamon SC. Electrophysiological characterization of voltage-gated currents in defined taste cell types of mice. J Neurosci 23: 2608–2617, 2003.
Miyoshi MA, Abe K, Emori Y. IP(3) receptor type 3 and PLCbeta2 are co-expressed with taste receptors T1R and T2R in rat taste bud cells. Chem Senses 26: 259–265, 2001.
Ninomiya Y, Higashi T, Katsukawa H, Mizukoshi T, Funakoshi M. Qualitative discrimination of gustatory stimuli in three different strains of mice. Brain Res 322: 83–92, 1984a.[CrossRef][Web of Science][Medline]
Ninomiya Y, Mizukoshi T, Higashi T, Katsukawa H, Funakoshi M. Gustatory neural responses in three different strains of mice. Brain Res 302: 305–314, 1984b.[CrossRef][Web of Science][Medline]
Ochocka AM, Pawelczyk T. Isozymes delta of phosphoinositide-specific phospholipase C and their role in signal transduction in the cell. Acta Biochim Pol 50: 1097–1110, 2003.[Web of Science][Medline]
Ogura T, Margolskee RF, Kinnamon SC. Taste receptor cell responses to the bitter stimulus denatonium involve Ca2+ influx via store-operated channels. J Neurophysiol 87: 3152–3155, 2002.
Richter TA, Caicedo A, Roper SD. Sour taste stimuli evoke Ca2+ and pH responses in mouse taste cells. J Physiol 547: 475–483, 2003.
Rodriguez I, Feinstein P, Mombaerts P. Variable patterns of axonal projections of sensory neurons in the mouse vomeronasal system. Cell 97: 199–208, 1999.[CrossRef][Web of Science][Medline]
Romanov RA, Rogachevskaja OA, Bystrova MF, Jiang P, Margolskee RF, Kolesnikov SS. Afferent neurotransmission mediated by hemichannels in mammalian taste cells. EMBO J 26: 657–667, 2007.[CrossRef][Web of Science][Medline]
Rossler P, Kroner C, Freitag J, Noe J, Breer H. Identification of a phospholipase C beta subtype in rat taste cells. Eur J Cell Biol 77: 253–261, 1998.[Web of Science][Medline]
Ruiz CJ, Wray K, Delay E, Margolskee RF, Kinnamon SC. Behavioral evidence for a role of alpha-gustducin in glutamate taste. Chem Senses 28: 573–579, 2003.
Tamura T, Hashimoto M, Aruga J, Konishi Y, Nakagawa M, Ohbayashi T, Shimada M, Mikoshiba K. Promoter structure and gene expression of the mouse inositol 1,4,5-trisphosphate receptor type 3 gene. Gene 275: 169–176, 2001.[CrossRef][Web of Science][Medline]
Tomchik SM, Berg S, Kim JW, Chaudhari N, Roper SD. Breadth of tuning and taste coding in mammalian taste buds. J Neurosci 27: 10840–10848, 2007.
Toyono T, Kataoka S, Seta Y, Toyoshima K. Expression of phospholipase C-{beta}4 in rat circumvallate taste buds. Chem Senses 30: i27–i28, 2005.
Wei JW, Chiang DH. Effects of calcium antagonists on KCl-evoked calcium uptake by rat cortical synaptosomes. Gen Pharmacol 17: 261–265, 1986.[Web of Science][Medline]
Yan W, Sunavala G, Rosenzweig S, Dasso M, Brand JG, Spielman AI. Bitter taste transduced by PLC-beta(2)-dependent rise in IP(3) and alpha-gustducin-dependent fall in cyclic nucleotides. Am J Physiol Cell Physiol 280: C742–C751, 2001.
Yang R, Crowley HH, Rock ME, Kinnamon JC. Taste cells with synapses in rat circumvallate papillae display SNAP-25-like immunoreactivity. J Comp Neurol 424: 205–215, 2000.[CrossRef][Web of Science][Medline]
Yee CL, Yang R, Bottger B, Finger TE, Kinnamon JC. "Type III" cells of rat taste buds: immunohistochemical and ultrastructural studies of neuron-specific enolase, protein gene product 9.5, and serotonin. J Comp Neurol 440: 97–108, 2001.[CrossRef][Web of Science][Medline]
Zhang Y, Hoon MA, Chandrashekar J, Mueller KL, Cook B, Wu D, Zuker CS, Ryba NJ. Coding of sweet, bitter, and umami tastes: different receptor cells sharing similar signaling pathways. Cell 112: 293–301, 2003.[CrossRef][Web of Science][Medline]
This article has been cited by other articles:
|
|
 |
|
  L. C. Geran and S. P. Travers Bitter-Responsive Gustatory Neurons in the Rat Parabrachial Nucleus J Neurophysiol, March 1, 2009; 101(3): 1598 - 1612. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hacker and K. F. Medler Mitochondrial Calcium Buffering Contributes to the Maintenance of Basal Calcium Levels in Mouse Taste Cells J Neurophysiol, October 1, 2008; 100(4): 2177 - 2191. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
