Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33101
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ABSTRACT |
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cAMP is a second messenger implicated in
sensory transduction for taste. The identity of adenylyl cyclase (AC)
in taste cells has not been explored. We have employed RT-PCR to
identify the AC isoforms present in taste cells and found that AC 4, 6, and 8 are expressed as mRNAs in taste tissue. These proteins are also expressed in a subset of taste cells as revealed by
immunohistochemistry. Alterations of cAMP concentrations are associated
with transduction of taste stimuli of several classes. The involvement
of particular ACs in this modulation has not been investigated. We
demonstrate that glutamate, which is a potent stimulus eliciting a
taste quality termed umami, causes a decrease in cAMP in
forskolin-treated taste cells. The potentiation of this response by
inosine monophosphate, the lack of response to D-glutamate,
and the lack of response to umami stimuli in nonsensory lingual
epithelium all suggest that the cAMP modulation represents umami taste
transduction. Because cAMP downregulation via ACs can be mediated
through Gi proteins, we examined the colocalization of
the detected ACs with G
i proteins and found that 66% of
AC8 immunopositive taste cells are also positive for gustducin, a
taste-specific G
i protein. Whether AC8 is directly
involved in signal transduction of umami taste remains to be established.
immunohistochemistry; glutamate; umami; taste transduction; gustducin
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INTRODUCTION |
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MEMBRANE-ASSOCIATED
ADENYLYL CYCLASES (AC) are enzymes that are central in the
regulation of intracellular cAMP levels. ACs, comprising a family of
nine isoforms (AC1-9), are integral membrane proteins, composed of
two cytosolic catalytic domains, C1 and C2, each preceded by six
transmembrane domains (16, 34). The catalytic domains are
highly conserved across all ACs and even across many guanylate cyclases
and are responsible for the synthesis of cAMP using ATP as a substrate.
Physiologically, G protein-coupled receptors (GPCRs) regulate ACs
through heterotrimeric G protein subunits, specifically the stimulatory
Gs, the inhibitory G
i, and/or certain
G
-subunits (9, 45). All AC isoforms can be
stimulated by G
s and by forskolin (FSK), a diterpene
commonly used in cAMP analysis. Inhibition by G
i is
limited to a subset of AC isoforms. Inhibitory G
i
subunits are able to overcome the FSK-mediated activation of AC1, 3, 5, 6, 8, and 9, presumably because FSK binds near the catalytic site,
distant from the G
i binding site (16, 55).
ACs also differ in their response to modulators such as
Ca2+ and calmodulin. Indeed, a unified classification of
ACs incorporates both such regulatory properties and their sequence similarities.
Taste buds are heterogeneous clusters of taste receptor cells, each of
which responds to subsets of taste stimuli and contains distinct
populations of cell signaling molecules. Modulation of cAMP has been
demonstrated in the sensory transduction of stimuli that elicit sweet
and bitter tastes (43, 52). However, the effect of
glutamate on cAMP modulation in taste tissue has not been described
previously. Glutamate elicits a taste quality termed "umami" that
is potentiated by the simultaneous presence of the monophosphates of
guanosine and inosine nucleotides. The potentiation is seen in
electrophysiological measurements of afferent nerve responses, as well
as in behavioral studies (12, 32, 38, 39, 51). Patch-clamp
recordings of rodent taste buds suggest that the electrophysiological
response to glutamate is mediated by a decrease in cAMP (5,
22). Candidate GPCRs for the sensory transduction of umami taste
have been proposed and include a truncated metabotropic glutamate
receptor, taste-mGluR4, and the heterodimeric receptor T1R1/T1R3
(10, 21, 31). Many metabotropic glutamate receptors are
known to couple to an inhibitory cAMP cascade (44). In
Chinese hamster ovarian (CHO) cells, the decrease in cAMP is thought to
be mediated by Gi and can overcome FSK-stimulated AC
activation. Taste-mGluR4 expressed in heterologous cells also exhibited
G
i-mediated inhibition of FSK-stimulated cAMP
(10), but signaling downstream of T1R1/T1R3 has not been established.
Gustducin is a G-subunit that is found at high concentration in a
subset of taste cells (28). Other G
-subunits
prominently expressed in taste cells include G
s and
G
i2 (20). The involvement of gustducin in
taste responses to many bitter and sweet compounds is supported through
a variety of biochemical, genetic, and electrophysiological analyses
(14, 24, 26). Recent behavioral experiments with knockout
mice suggest that gustducin may also be involved in umami taste
transduction (17, 37).
In early work, AC activity was demonstrated in taste tissue (42, 43), although which AC isoforms are expressed remained to be identified. The balance between the activities of ACs and phosphodiesterases (PDEs) determines the level of cAMP in cells. Several types of PDE have been identified in taste tissue, and taste tissue exhibits high basal PDE activity (36). The molecular identities of taste-expressed PDEs have been difficult to confirm, although recent studies have shown the presence of PDE3 (28) and two isoforms of PDE 1A (29). Pharmacological studies also suggest the presence of PDE4 in taste tissue (19). Whether the cAMP modulation in response to taste stimuli is achieved by regulation of AC or of PDE or both has not been studied in detail.
We employed RT-PCR and immunohistochemistry to identify the ACs in
taste tissue and demonstrate the presence of AC4, AC6, and AC8 in taste
receptor cells. We also demonstrated modulation of cAMP in rat taste
epithelium ex vivo by measuring cAMP levels in the presence of FSK, a
broad AC activator, and IBMX, a broad PDE inhibitor. We further tested
whether cAMP levels are altered when taste buds are stimulated with
taste-effective concentrations of glutamate. We found that glutamate
decreased cAMP. Downregulation of cAMP via ACs can be mediated through
Gi proteins. We examined the colocalization of detected
ACs with G
i proteins and found that 66% of
AC8-immunopositive taste cells are also positive for gustducin, a G
taste-specific protein, which belongs to a Gi/o/t/z subfamily.
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EXPERIMENTAL PROCEDURES |
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RT-PCR.
RNA was isolated from brain, circumvallate (CV), and foliate taste
papillae, nontaste lingual tissue, as well as from enzymatically delaminated taste and nontaste epithelia (15).
Poly(A+)RNA was isolated by affinity chromatography on oligo (dT)
cellulose (FastTrack kit, Invitrogen), whereas total RNA was isolated
by Nanoprep kit (Stratagene, La Jolla, CA). These RNAs were used as
template for cDNA synthesis using Thermoscript RT or Superscript II
(Life Technologies, Bethesda, MD). Degenerate primers were designed
from the most conserved region of ACs, corresponding to the sequences
LGDCYYC (5'-T/CTIGGIGAC/TTGC/TTACTACTG-3') and KIKTIG
(5'-C/G,T/CICCIATG/AGTC/TTTG/AATCTT-3') in the large cytoplasmic loop
and COOH-terminal domain, respectively. The PCR reactions were carried
out using a "touch-up" protocol with annealing temperatures rising
at 1°C/cycle from 37-47°C. RT-PCR products were concentrated
by ethanol precipitation before digestion with XbaI and StuI
(Life Technologies). Specific primers for AC2, AC3, AC4, AC5, AC6, AC7,
AC8, and AC9 and the amplification conditions were as previously
described (3). For amplification of AC1, primers were
designed in the region conserved between mouse and human cDNA sequences
(Table 1).
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Immunohistochemistry.
Frozen sections (30 µm) of rat CV (previously fixed in 4%
paraformaldehyde and cryoprotected in 30% sucrose overnight at 4°C) were blocked in 5% BSA, 2% normal donkey serum, and 0.025% Triton X
in phosphate-buffered saline (PBS) for 1 h at room temperature. Before blocking, endogenous peroxidase activity was quenched with 30%
H2O2 for 30 min at 20°C. Sections were
incubated overnight at 20°C with 1:300 diluted polyclonal antibodies
against AC3, AC4, AC5/6, or AC8 (all from Santa Cruz Biotechnology,
Santa Cruz, CA). The closely related isoforms AC5 and AC6 are detected
by a common antibody (designated AC5/6). Goat anti-rabbit
horseradish-peroxidase-labeled secondary antibody (1:1,000; Sigma) was
used for AC3, AC4, and AC5/6, whereas donkey anti-goat antibody
conjugated to Alexa 488 (1:1,000; Molecular Probes) was used to detect
AC8. The signal for AC3, AC4, and AC5/6 was amplified and detected with
tyramide-cyanine 3 (Perkin-Elmer, Boston, MA). For double
immunostaining, rabbit anti-gustducin (1:4,000), mouse
anti-Gi2 (1:1,000), and goat anti-G
i/o/t/z (1:1,000) were used, all from Santa Cruz
Biotechnology. Secondary antibodies were donkey anti-rabbit conjugated
to Alexa 594 for gustducin (1:1,500), donkey anti-mouse conjugated to
Alexa 488 (1:1,000), and rat anti-mouse Cy3 antibody (1:150, Jackson ImmunoResearch), both for G
i2 and donkey anti-goat
conjugated to Alexa 488 (1:1,000) for G
i-total, all from
Molecular Probes (Eugene, OR) unless otherwise stated. The
G
i-total antibody (Gi/o/t/z) detects all
members of the G
i family. Images were obtained on an
Olympus Fluoview confocal microscope. We confirmed using immunoblotting that each of the anti-ACs reacted against an antigen of the appropriate size in brain membrane extracts.
cAMP measurements. Male Sprague-Dawley rats 6- to 8-wk-old were used in all experiments. Epithelial sheets from CV papillae and adjacent nonsensory lingual surface were prepared by subepithelial injection of a protease cocktail containing 1 mg/ml collagenase D, 2.5 mg/ml dispase II, and 1 mg/ml trypsin inhibitor (15). Taste epithelial sheets containing taste buds were trimmed to remove most of the nontaste, surrounding epithelium, and von Ebner's glands, as described in detail (42). The sheets were then cut in the midline to yield equal left and right halves, which served as paired control and treated samples (Fig. 3A). Pairs of epithelial sheets containing taste buds were treated with FSK (1 or 10 µM) in Tyrode's buffer for 9.5 min at 30°C, followed by 30 s of either Tyrode (control) or taste stimuli [glutamate and/or inosine 5'-monophosphate (IMP)], both in the continuing presence of FSK. The reaction was terminated and cAMP was extracted from tissue using 70% perchloric acid as described (19). cAMP was then quantified using an enzyme immunoassay kit (Amersham Pharmacia). In Fig. 4, cAMP concentration in taste-stimulated epithelium is expressed as a fraction of the cAMP in the paired control taste epithelial sheet. Protein levels in the acid-precipitated tissue were measured using a NanoOrange Protein Quantitation kit (Molecular Probes) and were used to normalize the absolute value of cAMP before comparing treated vs. control samples. As an additional control, we used nontaste epithelial sheets (devoid of taste buds) and measured cAMP, with and without FSK.
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RESULTS |
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We designed degenerate PCR primers to two conserved sequences
(LGDCYYC and KIKTIG) within the C1 and C2 domains of types AC1-9 from mouse and rat. The RT-PCR product from brain RNA yielded a
heterogeneous product that included an 1,720-bp band (the size expected for AC1 and 8), a broad band of 1,790-1,836 bp
(AC2-7), and a band at 2,010 bp (AC9). This is consistent with the
known presence of all ACs in the brain (27). Poly (A) RNA
from CV and foliate papillae was reverse transcribed, used in a
parallel amplification reaction, and yielded bands in the 1,721- to
1,836-bp range (Fig. 1A). To
identify ACs present in taste receptor cells, we used diagnostic
restriction digestion of the degenerate PCR product. When the taste
amplification product was cut with XbaI, a diagnostic band
of 1,356 bp was detected (Fig. 1B, arrowhead). A unique
restriction site for XbaI is found only in AC4,
demonstrating the presence of this AC in taste tissue. The presence of
DNA not digested by XbaI indicated the presence of
additional ACs. A similar analysis with StuI digestion suggested the
presence of AC3/AC8 (~1,575 bp) and AC5/AC6 (~1,550 bp).
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Taste papillae contain many cell types in addition to taste receptor cells. To minimize the presence of extraneous cell types, we extracted RNA from trimmed taste epithelium, delaminated from underlying lingual tissue. This RNA was reverse transcribed and used in RT-PCR. Using primer pairs specific for each AC isoform, we confirmed the presence of all nine ACs in brain RNA. By contrast, RNA from CV epithelium consistently revealed strong RT-PCR bands for AC4, AC6, and AC8. RT-PCR products corresponding to AC2, AC5, and AC9 were occasionally detected in some taste samples, whereas RT-PCR products for AC1, AC3, and AC7 could not be detected.
RT-PCR results suggested that ACs are present in taste cells. Next, we
wanted to confirm the presence of AC isoforms in taste cells at the
protein level. We carried out immunohistochemical analysis of the
prominently detected isoforms, AC4, AC5/6, and AC8, as well as AC3,
which was seen in RT-PCR from taste papillae (but not from delaminated
epithelium). AC3 immunoreactivity was confined to the
subepithelial-stroma and probably represents nerve fibers based on the
elongate profiles visible (Fig.
2A). No immunoreactivity for
AC3 was detected in taste buds, in agreement with our RT-PCR results
with delaminated epithelium (Fig. 1C). Immunohistochemistry with an antibody specific for AC4 revealed that a small subset of
spindle-shaped taste receptor cells express this isoform. The majority
of taste buds in each section contained two to five stained cells in
our 30-µm sections. Antibodies specific for the closely related
sequences, AC5 and AC6, and for AC8 similarly showed subsets of taste
receptor cells stained (Fig. 2, C-F).
Although immunostaining for AC8 was readily detected using fluorescent
secondary antibody, the signal for AC4 and AC5/6 required a
tyramide-based amplification for stronger visualization. In all
experiments, control sections processed in parallel showed no signal if
primary AC-specific antibodies were omitted (Fig. 2, G and
H) or if such antibodies were preincubated with the
immunogenic peptide (not shown). The cells immunopositive for AC4 and
for AC5/6 were spindle-shaped and slender. Cells immunoreactive for AC8
were broad and had rounded large nuclei (Fig. 2F,
arrowhead). Morphologically distinct taste receptor cell types have
been observed to express distinct markers (53). Although
we have not conducted detailed morphometric analyses, our results
suggest that the various AC isoforms may exist in different taste
receptor cell types.
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Taste cells are functionally heterogeneous and may contain transduction
components for responding to different sets of stimuli. Consequently,
when taste tissue is disrupted for biochemical analyses, nonphysiological combinations of G proteins (which are found in both
membrane and soluble compartments), receptors, and effector enzymes may
yield responses not found in intact tissue. Thus we stimulated intact
taste cells before measuring cAMP. We measured cAMP in isolated
epithelium of the CV papillae and also in the adjacent nonsensory
epithelium (devoid of taste cells). Basal cAMP level in CV epithelium
was 20.5 ± 3.8 fmol/µg protein (n = 8),
consistent with previous experiments on unstimulated CV epithelia
(19, 42). Isolated epithelium from CV papillae was cut in
half along the midline (Fig.
3A), and pieces were treated for 9.5 min with either FSK, to raise baseline cAMP levels, or with
Tyrode's (control). In taste epithelium, 1 and 10 µM FSK increased
cAMP approximately threefold (to 59.9 ± 7.2 fmol/µg protein,
n = 6) and 10-fold (to 203 ± 41.8 fmol/µg
protein, n = 5), respectively (Fig. 3B).
When taste epithelia were incubated with both IBMX and FSK, cAMP levels
increased to 1,830 ± 611 fmol/µg protein. Such an increase
confirms the presence of high basal PDE activity in CV epithelium, as
previously suggested (36).
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A candidate taste receptor for umami stimuli, taste mGluR4, showed
glutamate-dependent cAMP decrease in FSK-pretreated cells (10). Other mGluRs also show similar coupling to an
inhibitory cAMP cascade (44). Hence, we measured cAMP in
native taste epithelia pretreated with FSK. After FSK pretreatment,
half of a CV epithelium (Fig. 3A) was stimulated with
glutamate for 0.5 min while the other half was not, and served as a
control. The range of glutamate concentrations used was based on the
known threshold for glutamate preference in rodent behavioral studies
(32). Within the range of glutamate concentrations tested
(1-20 mM), the response was greatest at 20 mM glutamate (cAMP
concentration was 65.7 ± 8.1% of that in paired control taste
epithelium treated with only 1 µM FSK; Fig.
4A). The
response was taste tissue specific, insofar as glutamate (at all tested
concentrations) did not induce a decrease in cAMP in nontaste
epithelium. cAMP levels upon glutamate treatment in nontaste epithelium
sheets were 117.6 ± 11.7% of paired control, nontaste epithelium
(n = 16, not significantly different from 100%).
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We also carried out stimulations on CV epithelium preincubated with 10 µM FSK. Again, 20 mM glutamate was found to trigger a decrease in cAMP levels to 75.2 ± 3.6% of FSK-treated control (n = 8; Fig. 4B). Higher concentrations of glutamate led to high variability in cAMP concentrations in both taste and nontaste samples, perhaps stemming from osmotic or other nonspecific effects. These were not pursued further.
In electrophysiological and behavioral assays, the taste response to glutamate is potentiated by the simultaneous presence of monophosphate nucleotides of inosine and guanosine. We measured cAMP in CV epithelium after stimulation with 20 mM glutamate, 0.5 mM IMP, and a mixture of both (Fig. 4B). Stimulation with IMP alone did not lead to an appreciable change in cAMP concentration. Compared with glutamate or IMP alone, the mixture caused a significantly greater decrease in cAMP. In contract, in nontaste epithelium stimulated with glutamate and IMP, cAMP levels were 94 ± 9% of paired control, nontaste samples (n = 9). The stereoisomer D-glutamate does not elicit umami taste. We found that 20 mM D-glutamate also did not modulate cAMP in CV epithelium at a concentration that produces a robust signal with L-glutamate (Fig. 4C).
Taste cells may mediate decreases of cAMP by regulating AC activity
through Gi proteins. In taste cells, the prominent
G
-subunits of the G
i family are G
i2
and G
gustducin. Hence, to determine whether ACs detected in taste
cells colocalize with G
i proteins, we used double immunohistochemistry.
Gustducin expression is well characterized in taste buds
(28). Because antibodies against gustducin, AC4, and AC5/6
were all raised in rabbit, we used antibodies against
Gi2 and against the entire class of G
i
proteins (i.e., G
i/o/t/z) in addition to anti-gustducin.
The mRNA for G
i2 is found in all gustducin-positive cells (1). We confirmed that this overlapping expression
also applies for G
i2 protein by double labeling
immunohistochemistry for gustducin and G
i2 (Fig. 5,
A-C). We also
confirmed that a common set of cells is labeled with antibodies against
G
i2 and G
i-total (Fig. 5,
D-F). Hence, we conclude that all three
antibodies against the G
i proteins detect the same cells
and can be used interchangeably.
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The expression profiles of AC4, AC5/6, and AC8 with Gi
proteins are presented in Fig. 6.
AC4-immunopositive cells did not colocalize with G
i2
(Fig. 6C). Although the overlay panel shows some yellow,
this appears to reflect separate cells that overlap in this section.
Note the distinct cellular shapes in the green and red panels where
each antigen is viewed separately (Fig. 6, A and
B). Taste cells strongly labeled with anti-AC5/6 did not overlap with G
i-total immunostaining. We noted that some
cells weakly stained with anti-AC5/6 did show robust signal for
G
i-total (Fig. 6, D-F). The
staining for AC5/6 was not sufficiently robust to quantify the overlap
of expression. In contrast, immunoreactivity for AC8 appeared to
colocalize with immunoreactivity for gustducin. To quantify this
colocalization, immunopositive cells that were well defined, with
visible nuclei, were counted in CVs from three rats. From each CV, 12 sections were randomly selected and three taste buds from one crypt of
each section were counted. Out of 200 cells positive for AC8, 66 ± 19% were also strongly positive for gustducin. Conversely, out of
203 cells immunopositive for gustducin, 57 ± 8% were also
strongly positive for AC8. In the CV, only 10-20% of all taste
cells are gustducin positive (7). Hence, the overlap
between gustducin and AC8 appears at a probability higher than chance.
We also observed a similar colocalization pattern of AC8 vs.
G
i2 (data not shown).
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DISCUSSION |
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Numerous AC isoforms exist that are subject to distinct regulatory controls and exhibit subtype-selective sensitivity toward physiological modulators. These ACs enable cells to fine tune cAMP levels and integrate signals between transduction pathways. Chemosensory systems are known to express several AC isoforms. The essential role of AC3 in olfactory neurons (49) and the presence of AC2 in vomeronasal neurons (4) have been described. The present study demonstrates for the first time that AC4, AC6, and AC8 are prominently expressed in taste tissue, both at the mRNA and protein level.
The second messenger, cAMP, is implicated in transduction of sweet and bitter taste qualities. For instance, sucrose increased cAMP levels in CV taste buds (42, 43), whereas denatonium and strychnine, two bitter substances, induced rapid reductions in cAMP (52). Electrophysiological studies in mammalian taste buds have suggested that modulation of cAMP levels may play a role in sour transduction (25). The role of cAMP in glutamate taste transduction is suggested by the damping of membrane currents when cAMP analogs were introduced into taste cells (23). We show here that rat taste cells undergo a decrease in cAMP levels when stimulated with 1-20 mM glutamate, a concentration range compatible with behavioral and afferent nerve responses in this species (41, 51). This decrease is taste specific because it is not present in nontaste epithelium. The potentiation of this response by IMP, the lack of response to D-glutamate, and the lack of response to umami stimuli in nonsensory epithelium all suggest that the cAMP modulation represents umami taste transduction.
The functional properties of AC4, AC6, and AC8 suggest that each could
readily be involved in transduction pathways in taste buds to signal
one or more taste qualities. In taste cells, increases of cAMP could be
mediated via AC4, AC6, or AC8, depending on their colocalization with
Gs. Furthermore, AC4 can be activated by G
-subunits (45, 46) to increase cAMP in a
Gs-independent manner downstream of Gi-,
Go-, or Gq-coupled receptors (47). Also, AC8, principally a brain-expressed isoform (40), can
be stimulated by calcium/calmodulin (50). The presence of
calcium-sensitive AC8 in taste cells suggests a means of resolving the
dual signaling pathways that have been noted for sweet transduction
(26). In gustducin-positive cells, the
-partners of
gustducin lead to activation of PLC
2, increased
IP3, and a consequent elevation of intracellular calcium.
In our experiments, AC8 colocalized with gustducin. This would explain
how saccharin increases both IP3 and cAMP
(30). AC8 is stimulated via capacitative calcium entry
(13), a mechanism that is also compatible with the
presence of a Trp channel (35) and capacitative calcium
currents in taste cells (33). Recently defined splicing
variants of AC8 are known to be differentially sensitive to such
regulation (8). However, our RT-PCR primers could not
discriminate between these types.
Decreases of cAMP have been noted in response to certain taste
stimuli, but attributing these to particular ACs is difficult. Direct
inhibition of AC4 by Gi2 proteins has not been
described; hence, it is not surprising that colocalization of AC4 and
G
i2 was not observed. In many tissues, the closely
related AC5 and AC6 are responsible for downregulation of cAMP. For
instance, AC6 is inhibited by low concentrations (<1 µM) of calcium
(11) and by G
i (54). We found
that taste cells that stained prominently for AC5/6 did not strongly
stain for G
i-subunits. However, AC6 is also inhibited by
certain G
combinations, notably
1
2 (2). Because the functional properties of G
1
2 are very similar to those of G
1
13 (6) and the latter dimer is present in
many taste cells (18), these G proteins may serve to
inhibit AC6. Although inhibitory regulation of AC8 has not been
examined, we postulate by analogy with the homologous AC1 that AC8
might be inhibited by G
-subunits released from Gi
proteins (48). The substantial overlap between AC8 and
both gustducin and G
i2 suggests that AC8 may be the
principal target for inhibitory regulation in taste cells. Whether such
inhibition arises from the G
or the G
-subunits remains an open question.
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ACKNOWLEDGEMENTS |
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We thank Laura Cooney for technical assistance with cAMP measurements, Elizabeth Pereira for assistance with immunohistochemistry, and Dr. Alejandro Caicedo for early help on confocal microscopy.
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FOOTNOTES |
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This work was supported by a grant from National Institutes of Health/NIDCD DC-03013 to N. Chaudhari.
Address for reprint requests and other correspondence: T. Abaffy, Dept. of Physiology and Biophysics (R430), Univ. of Miami School of Medicine, 1600 NW 10th Ave., Miami, FL 33136 (E-mail: tabaffy{at}newssun.med.miami.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 26, 2003;10.1152/ajpcell.00556.2002
Received 27 November 2002; accepted in final form 11 February 2003.
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