Introduction
Taste is essential to the survival of the animals and to themaintenance of the quality of life. Most mammals feels sweet, bitter, sour, salty and umami tastes[1]. Tongue has four type papillae : filiform, fungiform (FU), foliate (FO) and circumvallate papilla (CV). Taste buds exist in three papillae except the filiform papilla. FU is distributed in the anterior two-thirds of the tongue, FO located on the posterior lateral edges of the tongue, and CV is aligned along the posterior border of the tongue[2].
Taste buds of the mammal were composed of three typesof spindle-shaped cells and one type cuboid stem cells. The taste signals, which are detected by the taste receptor cells, are delivered into the brain along a taste pathway[3].
Type 1 taste bud cells are the most abundant cells in tastebuds. Type 1 taste bud cells have large granules of 100-400nm diameters and irregular shape of the nucleus[4]. Type 1 taste bud cells express glutamate aspartate transporter (GLAST), indicating that they may be involved in glutamate uptake[5]. The renal outer medullary potassium channels (ROMK) is also expressed in type 1 taste bud cells. A potassium channel may be involved in potassium homeostasis [6], and it would be involved in salty taste transduction[7]. Type 1 taste cells would be considered as glial cells in neural tissues, and they were called supporting cells.
Type 2 taste bud cells usually located in edge of the tastebuds, and are less than 20% of taste bud cells. Type 2 taste bud cells did not have the conventional synapse structures[8], and express specific taste G protein linked receptors for sweet, bitter and/or umami tastes[9]. Because they serves to detect taste substances with receptors, they are called receptor cells,[10].
Type 3 taste bud cells possess only 5-7% in taste buds cellnumbers. Type 3 taste bud cells are the only cells that they have the synapses between primary afferent gustatory nerves. This cells are called presynaptic cells[11]. Recently, it was reported that they might involve in sour taste transduction [12].
Taste receptors are the kinds of G-protein coupled receptors(GPCRs), GPCRs are the largest family of receptors that are present on cell membrane proteins belong to group across the cell membrane seven times. GPCRs act indirectly through the process of activating the G protein to regulate the activity of cell membrane target protein. Taste receptors activate the phospholipase C (PLC) and the resulting production of inositol trisphosphate (IP3) induced the release of calcium ions from the intracellular calcium ion storage and the inflow of calcium ions by opening of transient receptor potential channel M5 (TrpM5). After all tastes signals are converted to depolarization, the increase of intracellular calcium ions elicit the release of neurotransmitters, then the signals can be passed to taste nerves[13].
Taste receptors can be divided into type 1 taste receptors(T1Rs) and type 2 taste receptors (T2Rs). Type 1 taste receptors consist of three kinds of protein, T1R1, T1R2, and T1R3 [14], T1R2 and T1R3 proteins make up heterodimer to form sweet taste receptor, T1R1 and T1R3 proteins do heterodimer to form umami receptor [9,15].
Type 2 taste receptor genes are known to exist the 35 kindsin mouse and 25 kinds in human[16,17]. Recently, it was discovered that taste receptors were expressed in other area apart from the oral cavity. Tizzano et al[18] reported that type 2 taste receptors were expressed in airway of genetically engineered mice and rat by the reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. Singh et al[19] reported that the type 2 taste receptors were expressed in the brain of rats by immunohistochemistry and RT-PCR. Kwoen[20] reported that taste receptors were expressed in the submandibular, sublingual, and lacrimal glands employing the RT-PCR. Also Nishijima et al[21] also reported that the existence of taste buds and their nerve fibers in the larynx of rats by immunohistochemical study.
Sweetness and umami selecting nutritional substances issensitive in anterior tongue, but bitterness restricting the intake of probable toxic substances is sensitive in posterior tongue.
Even the sensitivity differences in different tongue areawere well known, however, the systemic study of the differences of expression levels of taste receptor genes has not been reported yet. In this study, the expression levels and expression patterns of the taste receptor genes in tongue papillae were examined by qPCR.
Materials and Methods
Animal
This study was approved (GWNU-2013-12) in Gangneung-Wonju National University Animal Experiment Ethics Commission, and was carried out and supervised by the Commission. DBA/2 male mice of 42-60 days old were used, because DBA/2 mice(Oriental Bio, Republic of Korea) are sensitive to bitter taste[22], and bitter taste receptors were well detected in our previous experiments[20].
Separation of mouse taste buds
After sacrificing the animal by cervical dislocation, tonguewas immediately excised. The isolated tissues were stored in HEPES buffered Tyrode solution (140 mM NaCl, 5 ml KCl, 1 mM CaCl2, 1mM MgCl26H2O, 10mM HEPES, 5mM glucose, 5mM pyruvate, pH7.4). For tissue sampling, the enzyme cocktail solution containing collagenase A (boehringer ingelheim, Germany, 1 mg / ml) and, dispase II (boehringer ingelheim, Germany, 0.25 mg / ml), trypsin inhibitor (1 mg / ml) in Ca2+free HEPES buffered Tyrode solution(140 mM NaCl, 5 ml KCl, 2 mM EGTA, 5 mM glucose, 10 mM HEPES, 5 mM pyruvate, pH 7.4) were injected beneath the papillae. After 30 minutes treatment at 37 ℃, the epithelial tissues including papillae were peeled off. CV and FO were dissected from the peeled off posterior tongue epithelium. FUs were collected about 300 each with glass micro-pipette from the peeled off anterior tongue epithelium.
RNA preparation
From the collected taste epithelial tissues, the RNA wasextracted with RNeasy Micro Kit (Qiagen, USA). RNA extraction procedure is RLT buffer including guanidine thiocyanate and β-mercaptoethanol 100: 1 ratio into a mixed solution mixed by centrifugation, and then 70% ethanol in a 1: 1 mix. After mounting it in the prepared column, and washed with RW1 buffer containing ethanol. DNase I incubation mix into a column placed in 15 minutes at room temperature. After washing the RW1 buffer containing ethanol, and again washed with RPE buffer. And RNA dried by centrifugation, washed with 80% ethanol, dried and put the RNase-free water to extract the RNA. RNA is prepared and stored in the next experiment at –20 ℃.
DNA synthesis
From extracted RNA, the ReverTra Ace® qPCR RT MasterMix with gDNA Remover (TOYOBO, Japan) was used to synthesize cDNA. cDNA is then denatured for 5 minutes at 65 ℃ by preparing RNA from 0.5μg. And DN Master Mix, Nuclear free water containing RNase inhibitor to denaturing RNA mix incubated for 5 minutes at 37 ℃. Reverse Transcriptase, RNase inhibitor, and then mixed with oligo dT primer and RT Master Mix containing the incubated for 15 minutes at 37 ℃ again. In 98 ℃, 5 minutes to stop the reaction by heating. The synthesized cDNA was kept until the next step at -20 ℃.
Primer
In this study, we used primers that used the RT-PCR in kwoen[20].
Quantitative polymerase chain reaction (qPCR)
100ng cDNA and primers (Table 1 and 2) that specific totaste receptor genes confirmed single PCR in Kwoen[20] were mixed KOD SYBR ® qPCR Mix (TOYOBO, Japan). After a denaturation at 95℃ with 3 seconds, process at 95 ℃ with 5 seconds, at 58 ℃ with 30 seconds, it is repeated 60 cycles. At 95℃ with 10 seconds, 65 ℃ at 95 ℃ to 5 ℃ interval undergo a 5 second process to draw a melt curve. All the process was repeated at least twice (type 1 taste receptor) or three times (type 2 taste receptors) to check reproducibility.
Reagents
All reagents were purchased from Sigma (U.S.A.) except specifically marked.
Data analysis
All results were calculated by the relative value to theglyceraldehyde-3-phosphate dehydrogenase (GAPDH), Statistical significance between each group was tested using the variance analysis (ANOVA test).
Results
Type 1 taste receptor
The expressions of type 1 taste receptors relative to that of the GAPDH in FU, FO, CV were different (Fig 1).
The expression of type 1 taste receptors relative to that of the GAPDH were 5.68 × 10-6 ~ 1.5 × 10-2 folds in FU, 3.0 × 10-6 ~ 1.17 × 10-4 folds in FO, and 4.55 × 10-6 ~ 7.18 × 10-4 folds in CV respectively.
It showed that three type 1 taste receptors were differentlyexpressed among papillae, but there was no significant (p = 0.153). In addition, there was no significant differences in the expression of type 1 taste receptors each disc (p = 0.861).
Type 2 taste receptor
The expressions of type 2 taste receptors relative to that of the GAPDH in FU, FO, CV were different (Fig 2).
The expressions of type 2 taste receptors relative to that of the GAPDH were 8.5 × 10-8 ~ 3.7 × 10-1 folds in FU, 2.0 × 10-9 ~ 2.0 folds in FO and 1.3 × 10-7 ~ 5.8 folds in CV, respectively (Fig 2).
The expression levels of type 2 taste receptors in FO andCV were higher than that in FU (Table 3). In all tested taste papillae, Tas2r108 and Tas2r137 were shown the most two highest expression levels (Table 3, Fig 2). Tas2r108 expression levels were 2.1 × 10-1 ~ 2.4 folds to that of GAPDH, Tas2r137 was expressed in a range of 5.6 × 10-2 ~ 5.8 folds to that of GAPDH.
The expression levels of type 2 taste receptors in tastepapillae were significantly different (p <0.05). However, the expression levels among papillae were not statistically different.
Discussion
Taste is chemical sensation felt mainly in the oral cavityand is detected by the taste buds of the tongue papillae. Tongue has 4 kinds of papillae which are filiform papilla, fungiform (FU), foliate (FO) and circumvallate papilla (CV)[2].
FU is present in anterior tongue, approximately the 0.5mmdiameter and the bell shaped. FU sends tastes signals to central nervous system through the chorda tympani nerves. FO is located parallel to the back edge of the tongue, next to the mandibular molar. The taste signals from FU passes through both the glossopharyngeal nerves and chorda tympani nerves. Human CV is present in a V-shape side of the root of the tongue, the diameter of 2-8mm. Murine CV is present on the center of posterior border of tongue. CV sends tastes signals only through the glossopharyngeal nerve[23].
The taste sensitivies are different from area by area oftongue. The posterior of the tongue is sensitive to bitter, the anterior of the tongue is sensitive to sweet, salty, sour.
Tastes is converted through sensory afferent gustatory frompapilla, gustatory nerve projected to the dorsal part of nucleus tractus solitaries (NTS). The thalamic taste area, VPMpc, projects to the primary taste cortex, which forms the rostral part of the frontal operculum and adjoining insula. The main gustatory information is projected to the cerebral cover parts of the prefrontal cortex and the temporal island cortex[24].
Type 1 taste receptors exist the 3 kinds in mammalsincluding mouse and human. Type 1 taste receptors of the 3 kinds form a heterodimer act receptors of different taste. The T1R2 and T1R3 heterodimer is taking action sweet receptors. Sweetness is evoked by nutrients of most carbohydrates. The T1R1 and T1R3 heterodimers act umami receptors. Umami taste is elicited also another nutrients of amino acid, glutamic acid[9,15,25]. Glutamate is rich components of fermented peptides, such as soy bean sauce, fish sauce, and cheese.
Type 2 taste receptors play the bitter taste receptor.Bitterness has a function that limits the intake of probable toxic molecules, such as various materials, including alkaloids. Type 2 taste receptors are reported 35 kinds in mouse and 25 kinds in human[16,17]. Type 2 taste receptor cells is characterized by expression of taste-specific G-protein, α-gustducin or PLCb2[17].
In principle, the PCR is repeated replication of the DNAtemplate using a DNA polymerase. The specific DNA polymerization is triggered by the specific reciprocal nucleotide sequence on both ends of the DNA template to be replicated. Double strands of the DNA template are separated and independent for each replication phase. Final products of PCR can be identified through separation by gel electrophoresis and dye staining. However, PCR can only check whether the desired DNA is present or not, it is very difficult to know how much does the target gene express.
Quantitative PCR is a method for measuring the amount of PCR product amplified in every PCR cycle. And In thisstudy, we used the SYBR green as a fluorescent tracer. Because the SYBR green can emit the fluorescence with any DNA bases, the primers for qPCR should be specific. The primers used in this experiments were very specific to every taste receptor genes and others[20]. Thus the qPCR results from this study were considered as the reflection of expression levels of those genes.
Result of the qPCR, FU, FO, CV were expressed 35 kindstype 2 taste receptor and three type 1 taste receptor. Tas1r3 is the component of both sweet and umami taste receptors, it would be expressed more than others, however, in this experiment Tas1r2 was expressed more. This result might be due to taste specificity of strain of mice used. DBA2 mice might be functionally Tas1r2 sweetness is much more important physiologically than the rich more than the expression used in the study was Tas1r3. Also DBA2 mice might be sensitive bitter taste but less sensitive umami taste. Because expression levels of taste receptor mRNA were 10-2~10-6 times to that of GAPDH, the possibilities are low, this may be experiment error. Most sweet stimuli were similar, nerve recordings of some sweet stimuli were significantly larger in the chorda tympani (CT) nerve than in the glossopharyngeal (NG) nerve[26]. However, analysis variance results did not show significant difference in the papilla. It may be due to small sample size.
In the case of type 2 taste receptor, FO and CV showedhigher expression levels than FU expression levels (Table 4, Fig 2 and 3). This result is reflected that FO and CV are distributed to posterior of tongue which more sensitive to bitter taste. In particular, the expression levels of Tas2r108 and Tas2r137 are frequently than the other type 2 taste receptors (Fig 2). If you check the relative expression of the GAPDH of type 2 taste receptor, while the majority of type 2 taste receptor is showing less expression than GAPDH (Fig 3). Tas2r108 and Tas2r137 are similar to GAPDH, or showed much expression (Fig 2). Ligands of Tas2r108 are denatonium, quinine, colchicine, diphenidol, caffeine, dapsone and Ligand of Tas2r137 is known as chloroquine[27,28]. Tas2r108 acts take a wide range of groups for detecting various bitter taste substances[29]. Tas2r108 is commonly expressed, but it is not known that many expressed Tas2r137, it is very interesting. The type 2 taste receptor, except for Tas2r108 and Tas2r137 can be considered some of the possibilities for a very small, but important physiologically. Type 2 taste receptor relative to their particular taste buds can only express a note of this expression is less likely. Unlike type 1 receptor, type 2 taste receptors are expressed in other organ as well as taste bud.
Distribution analysis results showed that the type 2 tastereceptors are a significant difference in the papilla. However, the expression between the taste receptors, between the papilla was confirmed that there are no significant differences. It may be due to small sample. Therefore, it is necessary to increase the number of sample used study.
Differences in taste sensitivity of the tongue would berelated to specific areas of the tongue taste threshold. Also, more expression of Tas2r108 is considered that Tas2r108 has a lot of ligand as it is known in the prior, more expression of Tas2r137 yet be studied.
Through qPCR it was able to determine the expressionpattern of the three groups taking taste papillae. Confirmed that 3 kinds of type 1 group and 35 kinds of type 2 group, but because not all of the expression in the papilla not make a significant difference, it is necessary to obtain a significant difference by additional research.
