Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Acetylcholine and Acetylcholine Receptors in Taste Receptor Cells
Department of Cell and Developmental Biology, University of Colorado Health Sciences Center at Fitzsimons, CO 80045, USA
Correspondence to be sent to: Tatsuya Ogura, e-mail: tatsuya.ogura{at}uchsc.edu
Key words: calcium imaging, immunohistochemistry, mudpuppy, neuromodulator, neurotransmitter, rodent
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Introduction |
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 Top Introduction AcknowledgementsReferences |
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Neuroactive substances play important roles as transmitters and neuromodulators. Although many of these substances and/or their receptors are known to be present in taste receptor cells and nerve fibers innervating taste buds, physiological functions of these substances are not well understood (Nagai et al., 1996
). Using Ca2+ imaging and immunocytochemical
techniques, we have examined the physiological responses of taste receptor cells to
acetylcholine (ACh), classified the types of ACh receptors, and determined the underlying
signaling mechanisms in taste receptor cells. Our results suggest that ACh may be
involved in cell-to-cell communication within the taste bud and in neuromodulation of
taste transduction mechanisms.
In Ca2+-imaging study, freshly isolated taste receptor cells were
loaded with Ca2+-sensitive fluorescent dye Fura-2 and intracellular
Ca2+ levels were measured ratiometrically. ACh induced increases in
intracellular Ca2+ levels ([Ca2+]i)
in taste receptor cells of mouse, rat and mudpuppy. The magnitude of the peak
Ca2+ response to ACh was concentration-dependent with half-maximum
responses around 1 µM. To determine which subtypes of ACh receptors and signaling
pathway were involved, we examined the effect of receptor antagonists and inhibitors for
selective pathway. Atropine (0.5 µM), a muscarinic ACh receptor antagonist, blocked
the ACh response, while D-tubocurarine (250 µM), a nicotinic ACh
receptor antagonist, had no effect. In addition, the phospholipase C (PLC) inhibitor
U73122
[GenBank]
(5 µM) and the Ca2+-ATPase inhibitor thapsigargin (1
µM), which depletes intracellular Ca2+ stores, blocked the ACh
responses. These results suggest that ACh binds to muscarinic ACh receptors, which
activates PLC, resulting in the production of IP3 and the subsequent release
of Ca2+ from the IP3-sensitive-intracellular stores. Since it
is known that binding of ACh to muscarinic receptor subtypes M1/M3/M5 activates the PLC
signaling pathways (Caulfield, 1993),
our data indicates the presence of at least one of these receptors in taste receptor
cells.
Additionally, we found that prolonged stimulation (>1 min) with ACh (10 µM)
induced a biphasic response with a transient followed by a sustained
[Ca2+]i increase. The sustained phase of the
[Ca2+]i increase was dependent on
Ca2+ influx as removal of extracellular Ca2+ eliminated
the response. Subsequently adding external Ca2+ induced increases in
[Ca2+]i, suggesting Ca2+ entry
through Ca2+-permeable channels. This is consistent with our previous
studies showing presence of Ca2+ store-operated channels (SOC) in taste
cells (Ogura, 2002;
Ogura et al., 2002). SOCs are
activated solely by store depletion without requirement of a receptor-mediated mechanism,
a mechanism also known as ‘capacitative calcium entry’. Thus it is possible
that the sustained part of the ACh-induced calcium response is mediated by
Ca2+ influx through SOCs.
In immunocytochemical study, sections containing rat circumvallate and foliate papillae were immunoreacted with an antiserum against the M1 subtype of muscarinic ACh receptors. Positive reaction was observed in many taste cells of each taste bud. In cross-sections of rat circumvallate papillae, roughly half of the taste cells were immunolabeled. No selective labeling was observed in control sections, in which primary antibody was omitted. Preabsorption with antigen significantly reduced the labeling. This result suggests that taste receptor cells express M1 subtype of ACh receptor. Thus ACh can bind to the M1 subtype of the muscarinic receptors and activate the PLC/IP3 pathway.
To study whether ACh is stored in synaptic vesicles in taste receptor cells and/or adjacent nerve fibers, we immunolabeled the vesicular ACh transporter (VAChT), a key element of ACh-containing vesicle in mouse taste tissue. A subset of taste receptor cells exhibited positive immunoreactivity to the antibody against VAChT. In addition, certain nerve fibers surrounding or within taste buds are positively reacted to antibodies against VAChT. These results suggest that taste receptor cells could release ACh for cell-to-cell communications among taste receptor cells and/or synaptic transmission from taste receptor cells to taste sensory fibers. The presence of VAChT in adjacent nerve fibers also reveals a possibility of cholinergic modulations of taste receptor cells via the muscarinic receptors.
Taken together, our results demonstrate ACh responses and its signaling pathway in
taste receptor cells. Since ACh increases [Ca2+]i
via PLC-mediated pathway, ACh may regulate taste responses by means of changing
[Ca2+]i levels or PLC signaling. It is known that
the PLC pathway mediates taste responses to bitter, sweet and umami substances
(Ogura et al., 1997, 2002;
Zhang et al., 2003). Further
experiments are needed to demonstrate these modulatory effects.
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Acknowledgements |
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TopIntroductionAcknowledgementsReferences |
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Supported by NIH/NIDCD DC 005140 to TO, DC00443 to W.L. and P30 DC04657 to Diego Restrepo of University Colorado Health Sciences Center.
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References |
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TopIntroductionAcknowledgementsReferences |
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Caulfield, M.P. (1993) Muscarinic receptors—characterization, coupling and function. Pharmacol. Ther., 58, 319–379.[CrossRef][Web of Science][Medline]
Nagai, T., Kim, D.J., Delay, R.J. and Roper, S.D. (1996) Neuromodulation of transduction and signal processing in the end organs of taste. Chem. Senses, 21, 353–365.
Ogura, T. (2002) Acetylcholine increases intracellular Ca2+ in taste cells via activation of muscarinic receptors. J. Neurophysiol., 87, 2643–2649.
Ogura, T., Mackay-Sim, A. and Kinnamon, S.C. (1997) Bitter taste transduction of denatonium in the mudpuppy, Necturus maculosus. J. Neurosci., 17, 3580–3587.
Ogura, T., Margolskee, R.F. and Kinnamon, S.C. (2002) Taste receptor cell responses to the bitter stimulus denatonium involve Ca2+ influx via store-operated channels. J. Neurophysiol., 87, 3152–3155.
Zhang, Y., Hoon, M.A., Chandrashekar, J., Mueller, K.L., Cook, B., Wu, D., Zuker, C.S. and Ryba, N.J. (2003) Coding of sweet, bitter, and umami tastes: different receptor cells sharing similar signaling pathways. Cell, 112, 293–301.[CrossRef][Web of Science][Medline]
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