Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
The Nitric Oxide–Cyclic GMP Cascade in Sugar Receptor Cells of the Blowfly, Phormia regina
1 Department of Applied Physics and Chemistry, The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan, 2 Department of Information Network Science, The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan, 3 Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto 606-8585, Japan and 4 Department of Sciences for Natural Environment, Faculty of Human Development, Kobe University, Kobe 657-8501, Japan
Correspondence to be sent to: Tadashi Nakamura, e-mail: tad{at}pc.uec.ac.jp
Key words: cyclic nucleotide gated channel, guanylyl cyclase, nitric oxide synthase, patch clamp, tip recording
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Introduction |
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 Top Introduction Pharmacological examination of...CNG conductance on the...Ca2+ shifts induced by...DiscussionAcknowledgementsReferences |
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It is well known that nitric oxide (NO) is produced by NO synthase (NOS) in postsynaptic nerves and diffuses through membranes into presynaptic nerves where NO activates soluble guanylyl cyclase (sGC) to produce cGMP, resulting in the feedback control of presynaptic nerve activity. However, in taste sensory systems, the NO–cGMP cascade might act differently. In the vertebrate taste receptors, evidence for expression of NOS (Kretz et al., 1998
;
Zaccone et al., 2002),
function of cGMP as a second messenger (Okada
et al., 1987;
Tonosaki and Funakoshi, 1988;
Krizhanovsky et al., 2000)
and the regulation of the cGMP level via NO (Rosenzweig et al., 1999) have been reported, which
suggest that NO could function for the signal transduction in the taste receptor cells.
In the blowfly, Phormia regina, there have been reports that suggest the
importance of cGMP in the taste transduction.
Wieczorek and Schweikl (1985)
reported a high concentration of cGMP and high activity of guanylyl cylclase in the taste
sensillum-rich region.
Amakawa et al. (1990)
reported that membrane permeable cGMP induces impulses from sugar receptor cells. If cGMP
works as a second messenger in the sugar receptor cells, it may be possible that cGMP is
produced by sGC activated by NO in the blowfly sugar receptor cells.
In this paper, we review recent progresses in our studies on the taste transduction
in P. regina. Our study includes analyses by the tip recording (Murata et al., 2004), patch clamping
(Satoh et al., 2003) and
Ca2+ monitoring in the isolated receptor cells (Murata et al., 2003). Throughout these studies,
animals were reared under 12 h light/12 h dark cycles.
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Pharmacological examination of the NO signaling system in sugar receptor cells |
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TopIntroductionPharmacological examination of...CNG conductance on the...Ca2+ shifts induced by...DiscussionAcknowledgementsReferences |
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We performed pharmacological investigation to test whether NO participates in the transduction of the sugar receptor cells using the tip recording method. Impulses were induced from taste receptor cells in the largest chemosensilla of animals aged 5–7 days after emergence with a stimulating solution (100 mM sucrose/10 mM NaCl solution) in a glass capillary electrode. The intensity of the taste response was represented by the number of impulses during 150–350 ms after the beginning of the stimulation. To address whether NO is involved in sugar-mediated activation of the neurons in these sensilla, we introduced NO scavenger or NOS inhibitor into these cells by incubating the tip of the chemosensilla with a solution of each reagent plus 0.03% deoxycholate (DOC) for 2 min followed by 5 min of recovery time (DOC method: Ozaki and Amakawa, 1992
). Taste
responses were recorded before and after the introduction of those reagents into the
cells. The ratio of the impulse numbers of the latter record to that of the former record
was calculated (= relative response) to indicate the effect of each reagent. We first examined if the sugar response decreased by reducing the NO in the cell. Reducing the intracellular NO was achieved by applying the NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO), by the DOC method. Relative response was 0.46 ± 0.09 (n = 8) with 10 mM PTIO. This clearly indicates that NO is involved as an activating factor in the transduction cascade of the sugar receptor cells. PTIO hardly affected the responses to salt or water.
Next we examined the effect of increasing NO in the sugar receptor. NO was
continuously released from an NO donor,
1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC 7),
dissolved in the solution inside the tip recording electrode. When we applied freshly
prepared 8 mM NOC 7 solution (in 20 mM MOPS, pH 6.6) to a sensillum, two kinds of
impulses with different amplitudes appeared. Following small impulses (0.56 ± 0.01
mV, n = 20), large impulses (0.84 ± 0.01 mV, n =
20) started to appear with ~15 s of latency. By comparison with standard impulses derived
from sugar, salt, water and ‘fifth’ receptor cells according to
Ozaki et al. (2003), the
small impulses were attributed to those derived from the water receptor cells whereas the
large impulses were attributed to those from sugar receptor cells. It is very likely that
NO induced the impulses from sugar receptor cells. The long latency for the impulses from
sugar receptor cells might be the time necessary for NO to penetrate the membrane and to
be concentrated until at a sufficient level to activate sGC in the sugar receptor
cells.
Using the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 250 µM), we examined if NOS is involved in the transduction pathway in the sugar receptor cells. This reagent specifically decreased the response to 100 mM sucrose giving relative response of 0.66 (n = 7). However, the same amount of the inactive enantiomer of the L-NAME, NG-nitro-D-arginine methyl ester (D-NAME), had no effect, giving average relative response of 0.94 (n = 7). These results indicate that the NOS is involved in the neuronal activation of the sugar receptor cells in response to sucrose.
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CNG conductance on the dendritic plasma membrane |
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TopIntroductionPharmacological examination of...CNG conductance on the...Ca2+ shifts induced by...DiscussionAcknowledgementsReferences |
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Following Murakami and Kijima (2000
), labella
were dissected from pupae aged 12–24 h before the emergence. The sensilla were cut
at their middle of the largest type and incubated in the fly Ringer solution. In 20 min,
the sensory processes grew out from the cut end of the sensilla. When
Ca2+ in the extracellular solution was reduced, tips of the processes
swelled into small spheres. Using patch electrodes containing Ca2+-free
solution, we obtained inside-out patch membranes from such swollen parts. We recorded the
membrane current under the ramp voltage from –70 to 70 mV to monitor the
I–V relationship of the membrane conductance. In a small number of
membrane patches, we observed the conductance increase in response to 1 mM cGMP applied
to the intracellular side of the patch membrane. Although these data are preliminary,
they indicate the presence of CNG conductance on the plasma membrane. |
Ca2+ shifts induced by taste stimuli in the isolated receptor cell |
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TopIntroductionPharmacological examination of...CNG conductance on the...Ca2+ shifts induced by...DiscussionAcknowledgementsReferences |
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We developed the cell culture protocol for the taste receptor cells (Murata et al., 2003
). Twenty labella of
pupae aged 4–5 days after the pupation were disrupted by the incubation with 0.4
mg/ml papain in nominally Ca2+-free solution for 40 min, then shaken with
a mixer at 1500 r.p.m. for 2 min. Obtained cell suspension was plated and cultured in
modified Leibovitz’s L-15 medium at 29°C. We found bipolar cells survived for
2–6 days in the culture, some of which extended long processes. By monitoring the
intracellular Ca2+ in these bipolar cells with the
Ca2+-sensitive fluorescent dye, fluo-3, we found that some cells were
responsive specifically to sucrose, NaCl or LiCl. The response to sucrose was dependent
on the Ca2+ in the extracellular solution. Therefore the taste responses
are likely to occur through the opening of cation channels that allow the influx of
Ca2+ into the cells. |
Discussion |
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TopIntroductionPharmacological examination of...CNG conductance on the...Ca2+ shifts induced by...DiscussionAcknowledgementsReferences |
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Our tip recording experiments demonstrated that NO works for the impulse-generation in the sugar receptors as well as that NO is produced by the stimulation with sugar, which suggests that it is produced and functions for the impulse-generation in the same sugar receptor cell. This result raises the possibility that NO activates sGC to produce cGMP that can function as a second messenger. This possibility is supported by our preliminary observation that the dendritic membranes of the receptor cells contain the conductance gated by cGMP (CNG conductance). Thus it is very likely that the sugar receptor cells contain the NO-cGMP cascade as a part of the signal transduction pathways.
It is noteworthy that the IP3 gated channels (Ozaki and Amakawa, 1992;
Koganezawa and Shimada, 2002) or the
ionotropic receptors (Murakami and Kijima,
2000) could contribute to the NOS activation by allowing Ca2+
entry into the sugar receptor cells. In fact, we have preliminarily observed the
transient increase of Ca2+ in the excited receptor cells. That
observation might reflect the preceding process of the NOS activation. However, CNG
channels or voltage gated channels could also be a route for the Ca2+.
The relationships between those mechanisms and the NO–cGMP cascade are to be
studied. The study may lead to elucidation of the overall transduction mechanism in the
sugar receptor cells.
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Acknowledgements |
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Present study was partly supported by PROBRAIN to T.N.
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References |
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