Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Expression of Phospholipase C-ß4 in Rat Circumvallate Taste Buds
Department of Oral Anatomy and Neurobiology, Kyushu Dental College, Kokurakita-ku, Kitakyushu 803-8580, Japan
Correspondence to be sent to: Takashi Toyono, e-mail: toyono{at}kyu-dent.ac.jp
Key words: mGluR1
, , phospholipase
C-ß, 4, taste bud, umami
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Introduction |
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 Top Introduction Materials and methodsResults and discussionReferences |
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The heteromer of T1R1 and T1R3 and taste-mGluR4 function as receptors for glutamate (umami) taste sensation (Chaudhari et al., 2000
;
Nelson et al., 2002).
Metabotropic glutamate receptor type 1 (mGluR1) is expressed in taste receptor
cells in rat gustatory papillae (Toyono et
al., 2003). It has been known that mGluR1 couples preferentially
to an -subunit of the Gq family, leading to activation of phospholipase
C-ß (PLC-ß) and the consequent mobilization of intracellular Ca2+
levels in the central nervous system (Hermans and
Challiss, 2001). The inositol tri-phosphate (IP3) pathway is
involved in taste transduction for glutamate in mouse fungiform papilla (Ninomiya et al., 2000). Applications of
glutamate and the mixture of GMP and glutamate to rat taste cells increase intracellular
Ca2+ levels (Lin et al.,
2003). Thus, mGluR1 may be a candidate for another type of umami
receptors because mGluR1 plays some roles in IP3 pathway and the
mobilization of intracellular Ca2+.
Recent studies have provided evidence that the members of Gq family,
Gq, G14, G15 and PLC-ß2, are expressed in rat taste buds
(Kusakabe et al., 1998;
Rössler et al., 1998).
Further, PLC-ß2 is known to co-expressed with IP3 type III receptor
(IP3R3) in rodent circumvallate taste buds (Clapp et al., 2001). PLC-ß2 generates
IP3, which then activates IP3R3 of intracellular
Ca2+ stores in taste cells. In this context, it is conceivable that there
may exist a similar signaling cascade via mGluR1 in umami taste sensation.
However, no mention has so far been made of the expression patterns of mGluR1 and
these signaling molecules in rat taste bud cells.
There are four different PLC-ß isoforms (PLC-ß1–4) that have been
cloned (Rhee and Bae, 1997). They are
all regulated by heterotrimeric G proteins and there is evidence suggesting that
different isoforms may be involved in a variety of signaling circuits. PLC-ß can be
activated by both the G subunits of the Gq family and by the ß
subunits generated by a number of different heterotrimeric G proteins. On the other hand,
PLC-ß4 can be activated by Gq but not by ß-subunits of G-proteins
(Jiang et al., 1994). The
major molecular cascade from mGluR1 to PLC-ß is considered to be
mGluR1–Gq–PLC– ß4 in Purkinje cells (Hirono et al., 2001). In view of these respects, we
deduced that PLC-ß4 might contribute the mGluR1-mediated signal transduction in taste
sensation. However, a search of the literatures fails to reveal the expression of
PLC-ß4 in rat taste tissues.
In the present study, we examined for the first time the expression patterns of
mGluR1 and taste signaling molecules, Gq and PLC-ß2 in rat
circumvallate papillae. In addition, we examined the expression PLC-ß4 mRNA and its
protein in gustatory papillae and taste buds by using reverse
transcription–polymerase chain reaction (RT–PCR) and
immunohistochemistry.
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Materials and methods |
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TopIntroductionMaterials and methodsResults and discussionReferences |
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The methods used in this study were approved by the Institutional Animal Care and Use Committee of Kyushu Dental College.
In situ hybridization
Adult SD rats were anesthetized with chloral hydrate (350 mg/kg) and transcardially
perfused with a fixative containing 4% paraformaldehyde (PFA) in 0.1 M phosphate
buffer (pH 7.3) for 15 min. The areas of circumvallate papillae were dissected out and
rinsed overnight with 0.1 M phosphate buffer containing 30% sucrose. Tissues were
then embedded in Tissue-Tek and snap-frozen in a dry ice-isopentane mixture. In
situ hybridization analysis for mGluR1 were performed as described previously
(Toyono et al., 2003).
Immunohistochemistry
The frozen sections were prepared in similar manners to as described above. Sections were washed in PBS, and incubated for 12 h at room temperature in a solution containing rabbit polyclonal antibody against rat PLC-ß4 (Santa Cruz Biotechnology) at a dilution of 1:1500. AlexaTM488-conjugated goat anti-rabbit IgG (1:200; Invitrogen) was used as a secondary antibody. Negative controls to immunofluorescence staining were performed by replacing the primary antibodies with PBS or by pre-incubating of primary antibodies with cognate peptides. After immunostaining for PLC-ß4, double-labelled experiments were performed with Alexa Fluor 546-labelled PLC-ß2 rabbit polyclonal antibody (Santa Cruz biotechnology). This labelled antibody was prepared with the Zenon Rabbit IgG Labeling Kits (Invitrogen).
After in situ hybridization, some sections were analyzed for co-expression
of mGluR1-mRNA and Gq-protein, or of mGluR1-mRNA and PLC-ß2-protein by using
immnohistochemistry.
RT–PCR
RT–PCR analyses for PLC-ß 4 were performed as
described previously (Toyono et al.,
2003). Primer sequences for the PCR were as follows:
PLC-ß 4, 5'-ATCGTGGCCCAGTATGACAAG-3' (forward)
and 5'-ATCTGCTGCATCTCCTTCGC-3' (reverse); product size, 590 bp. PCR
amplifications were performed under the following conditions: 94°C for 30 s, 55°C
for 1 min, 72°C for 1 min for a total of 40 cycles and elongation step at 72°C
for 10 min for PLC-ß 4 and GAPDH.
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Results and discussion |
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TopIntroductionMaterials and methodsResults and discussionReferences |
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The expression patterns of mGluR1 and signaling molecules were examined by in situ hybridization and immunohistochemistry in rat circumvallate taste buds. A subset of mGluR1-expressing taste bud cells co-expressed PLC-ß2. According to the study by Zhang et al. (2003
),
PLC-ß2 deficient mice abolished sweet, amino acid and bitter taste perception. Their
study suggested that mGluR1 might play some roles in umami taste-signaling
cascades through PLC-ß2. We also examined the expression of Gq in
mGluR1 expressing cells in rat taste bud cells. Almost all mGluR1
expressing cells co-expressed Gq. Further, double immunolabeling experiments for
Gq and PLC-ß2 showed that almost all PLC-ß2 expressing cells co-expressed
Gq. In their study on the group I metabotropic glutamate receptors,
Hermans and Challiss (2001), found
that mGluR1 couples preferentially to an subunit of the Gq family,
leading to activation of PLC-ß in the central nervous system. Taken together with our
results it seems that, as well as the central nervous system, mGluR1 may couple
with Gq which consequently, may activate PLC-ß2 in umami taste transduction in
taste bud cells.
RT–PCR assay showed that PLC-ß4 -mRNA expressed in circumvallate papillae
(Figure
1A). In fungiform, foliate and
circumvallate papillae, the antibody against PLC-ß4 gave labelling of the subset of
taste bud cells and intragemmal and subgemmal nerve fibers (Figure
1B). Double labeled experiments showed
that a subset of PLC-ß4 expressing cells also co-expressed PLC-ß2 (Figure
1C–E). In the central nervous
system of the mouse, despite the existence of four PLC-ß isoforms
(PLC-ß1–4), only one or two of them is expressed in each neuron and glial cell
(Watanabe et al., 1998).
PLC-ß3 and PLC-ß4 are major isoforms with lower levels of PLC-ß1 in Purkinje
cells. Similar to the results obtained from mouse central nervous system, PLC-ß2 and
PLC-ß4 are expressed in a subset of taste bud cells and these isoforms may form a
functional IP3 signaling cascade, playing some roles in the taste signal
transductions. PLC-ß4 is known to work through mGluR1 in the mouse cerebellum, and
PLC-ß4-deficient mouse is reported to show ataxia (Kim et al., 1997). A clue to understand the role of
PLC-ß4 in taste transduction may be gain from the analysis of the PLC-ß4-null
mouse.
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TopIntroductionMaterials and methodsResults and discussionReferences |
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