Chem. Senses 27: 673-680,
2002
© Oxford University Press 2002
Two Second Messengers Mediate Amino Acid Responses in Olfactory Sensory Neurons of the Salamander, Necturus maculosus
Department of Biology, University of Vermont, Burlington, VT 05405, USA 1 Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA
Correspondence to be sent to: Dr Vincent E. Dionne, Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA. e-mail: vdionne{at}bu.edu
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Abstract |
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Odor transduction mediated by the adenylyl cyclase/cAMP pathway has been well studied, but it is still uncertain whether this pathway mediates the transduction of all odors in vertebrates. We isolated olfactory sensory neurons from the salamander Necturus maculosus and used calcium imaging with the indicator dye fura-2 to examine olfactory responses elicited by amino acids. The properties of approximately two-thirds of the odor responses suggested they were mediated by the adenylyl cyclase/cAMP pathway, but one-third of the responses were not mimicked by cAMP analogs nor blocked by inhibition of adenylyl cyclase, suggesting that these odor responses were mediated differently. Responses that were unaffected by inhibition of adenylyl cyclase were blocked by neomycin, an inhibitor of phospholipase C, implying that they were transduced by activation of phospholipase C. Some cells which responded to more than one amino acid appeared to employ both pathways, but each was used to transduce different odors. In addition, many responses that were mediated by the adenylyl cyclase/cAMP pathway were enhanced following inhibition of phospholipase C, suggesting that the phospholipase C pathway has a role not only in odor transduction, but also in the modulation of olfactory responses.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Odor responses are transduced by G-protein-coupled olfactory receptor proteins (ORs) that regulate intracellular biochemical pathways in olfactory sensory neurons (OSNs) (Schild and Restrepo, 1998
). In addition, odor responses can be modulated
through the actions of G-protein-coupled receptors, for example by neuroactive
compounds such as hormones (Kawai et
al., 1999; Eisthen et
al., 2000;
Wirsig-Wiechmann et al.,
2001). In invertebrate OSNs, at least two different intracellular
pathways mediate odor transduction; one uses adenylyl cyclase (AC) and its
product cyclic adenosine monophosphate (cAMP)
(Michel and Ache, 1992), the
other phospholipase C (PLC) and its product inositol 1,4,5-trisphosphate
(IP3) (Fadool and Ache,
1992). By comparison, odor transduction in vertebrate OSNs is
generally thought to be mediated by the AC/cAMP pathway alone
(Gold, 1999). Although PLC and
its product IP3 have been implicated in odor transduction in
vertebrates such as fish (Huque and Bruch,
1986; Restrepo et al.,
1990,
1993;
Miyamoto et al.,
1992), amphibians
(Kashiwayanagi, 1996) and
mammals (Boekhoff et al.,
1990; Rawson et al.,
1997; Lischka et al.,
1999), the activation of PLC in response to odor may be indirect,
with modulation of the odor response rather than transduction as its
consequence. A critical test of this issue has been difficult. The goal of
this study was to examine how odor responses in the salamander, Necturus
maculosus, depend on the AC/cAMP and PLC pathways. Necturus is a
neotenic amphibian that reaches lengths of 35-50 cm as an adult and has cells
that are considerably larger than those found in mammals or other
vertebrates. In most cases, odors produce depolarizing receptor potentials that open voltage-gated Na+ and Ca2+ channels to elicit action potentials. In Necturus voltage-gated Ca2+ channels of OSNs are located on the dendrite and cell body, allowing Ca2+ influx through these channels to elevate intra-cellular [Ca2+] throughout the cell. In addition, cytoplasmic [Ca2+] can rise in OSNs when cAMP-gated cation channels in ciliary membranes or IP3-gated cation channels are opened. As a result, Ca2+ is an effective reporter of depolarizing odor responses.
Using imaging techniques with the Ca2+-sensitive dye, fura-2, and OSNs isolated from Necturus, we find evidence that both the AC/cAMP and PLC pathways are used to transduce odor responses. In addition, our data indicate that these pathways can interact and that the PLC pathway can modulate the activity of the other.
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Materials and methods |
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Adult N. maculosus were obtained from commercial vendors and housed in aerated aquarium tanks at 10°C. The nasal tissues were removed by blunt dissection from animals that had been chilled on ice and decapitated. Olfactory sensory neurons were dissociated from the tissue without proteolytic enzymes as described previously (Dionne, 1992
). Briefly, the olfactory epithelium was peeled from
underlying connective tissue and cut into small pieces in amphibian
physiological saline (APS). The pieces were then incubated in a low
[Ca2+] saline for 18 min and placed in normal extracellular saline
with DNase for 10 min, then gently swirled. After swirling and allowing 10-30
s for debris to settle, isolated OSNs could be found in the supernatant. The
supernatant was plated onto Cell-Tak (Collaborative Biochemical Products) or
concanavalin-A-coated dishes, where the cells were allowed 20 min to settle
and adhere to the substrate. The saline was then replaced with fura-2 loading
solution containing 5 µM fura-2AM plus 0.02% pluronic F-127 for 10-30 min,
after which the cells were washed with APS (10 min) and used for
experimentation. Loaded cells were viewed with a compound microscope (either a
Nikon Diaphot or an Olympus BX) and imaged through a x40 objective. The
chamber was continuously washed with APS during recording. Stimuli were
dissolved in APS and delivered as a 10-30 s pulse with a Warner fast step
perfusion system. A cooled CCD camera (Astrocam Model TE3/A/S) was used to
collect 200 ms paired images at 350 and 380 nm wavelengths at 4 s intervals.
Differential imaging was controlled with a Spectramaster imaging system using
Merlin software (Life Science Resources, Cambridge, UK) to calculate image
ratios, calibrate and measure calcium concentration and display images both
on- and off-line. The absolute Ca2+ concentrations are estimates;
all conclusions about odor responses are based on relative changes.
Necturus OSNs respond to amino acids, bile salts and various other
soluble compounds as odorants. Twelve amino acids were used as chemical
stimuli in these experiments (Table
1). All amino acids were the L-form, dissolved in APS and applied
at concentrations from 10 µM to 10 mM. Cells were also stimulated with the
phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), several
analogs of cAMP [8-bromo-adenosine 3',5'-cyclic monophosphate
(8-br-cAMP); 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic
monophosphate (8-cpt-cAMP); N6,2'-O-dibutyryladenosine
3',5'-cyclic monophosphate (db-cAMP)] and the forskolin analog
L-858051
[7-deacetyl-7-[O-(N-methylpiperazino)-
-butyryl]-forskolin] to
activate adenylyl cyclase. In some experiments cells were exposed to an
adenylyl cyclase inhibitor,
cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A),
or a phospholipase inhibitor, neomycin. The pH of all solutions was adjusted
to 7.2 with NaOH, except for choline-APS where Tris base was used. Fura-2AM
and pluronic F-127 were purchased from Molecular Probes. IBMX, MDL-12,330A and
L-858051 were purchased from Calbiochem-Novabiochem Corp., as were the cAMP
analogs. All other chemicals including neomycin sulfate and the amino acids
were purchased from Sigma Chemical Corp.
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Extracellular bath solutions were as follows. APS contained (in mM): NaCl, 112; HEPES [N-(2-hydroxyethyl)-piperazine-N'(2-ethanesulfonic acid)], 3; KCl, 2; CaCl2, 8; glucose 5. K-APS contained (in mM): NaCl, 12; HEPES, 3; KCl, 102; CaCl2, 8; glucose, 5.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The dissociated OSNs selected for testing appeared to be intact except for their axons, which had been severed; the somata were ovoid and the dendritic endings each held a tuft of cilia. Dendrites varied in length from
15 to
100 µm. OSNs were deemed viable if their resting intracellular
[Ca2+] was in the range of 20-100 nM before testing. Cells were
tested first with a puff of K-APS to depolarize them and verify an ability to
generate a transient rise in intracellular [Ca2+]; unresponsive
cells were discarded. Chemosensitivity was tested by applying one or more
amino acids individually. Although 12 amino acids were used in this study, not
all cells were tested with all stimuli. When a cell was seen to respond to one
or more odorants, it was treated with and/or tested with pharmacological
agents to examine the properties of the intracellular pathways that mediated
the odor responses. These results are based on recordings from >330
OSNs. Seventy-six OSNs responded to at least one amino acid with an increase in intracellular [Ca2+] (Figure 1). Typically, responses rose quickly to a peak concentration, returned to baseline with washing and were reproducible (Figure 2A). For most cells used in this study, only one amino acid was identified that elicited a response; this is unlikely to reflect the full range of the cells' chemosensitivity because most testing was stopped once an adequate stimulus was found. Fourteen of 41 OSNs that were tested with multiple amino acids responded to two or more compounds, but no cell tested with multiple amino acids responded to all the test stimuli. Eight OSNs showed a decrease in [Ca2+] (Figure 2B) rather than an increase in response to a stimulus; most of these cells (7/8) also showed an increase in [Ca2+] in response to other stimuli. Five different amino acids elicited [Ca2+] decreases (Table 1) and all but one (lysine) also elicited [Ca2+] increases in other cells. [Ca2+] decreases were difficult to characterize because of their small headroom (the resting [Ca2+] was already low) and their low incidence; they will not be discussed further. Finally, many apparently healthy cells responded only to K-APS but not to any of the test stimuli, suggesting that an adequate stimulus for those cells had not been included or that their responses did not involve changes in intracellular Ca2+.
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Involvement of the AC/cAMP pathway
OSNs that responded to one or more amino acids were used to test the involvement of the AC/cAMP pathway in the response.
Increased [cAMP]
In 37 of 60 cells (62%) that responded with a [Ca2+] increase to
one or more amino acids, the Ca2+ response was mimicked by
elevating intracellular cAMP or by applying a cAMP analog
(Figure 2A); however, the
responses in 23 of the 60 cells (38%) could not be mimicked this way.
Three membrane-permeant analogs of cAMP along with IBMX were used to
stimulate cAMP-dependent responses
(Firestein et al.,
1991). Db-cAMP, 8-br-cAMP and 8-cpt-cAMP were tested at
concentrations of 10-1000 µM. All three compounds were able to elicit
Ca2+ responses, but none were as effective as IBMX. IBMX was also
tested at concentrations of 10-1000 µM. In most cells the Ca2+
responses elicited by IBMX did not increase monotonically with concentration.
Intermediate concentrations of IBMX (50 and 100 µM) frequently elicited the
largest increases in [Ca2+]
(Figure 3A), but the most
potent concentration of IBMX varied among individual OSNs. The cause of the
non-linear dose—response relation was unclear. Although the magnitude of
the Ca2+ response showed adaptation by decreasing with repeated
applications of IBMX, the non-monotonic dose—response relation did not
appear to be a consequence of adaptation. The Ca2+ responses
induced by IBMX required bath Ca2+, indicating that they were
mediated by an increase in the Ca2+ permeability of the membrane
(Figure 3B). Similarly, the
amino acid-induced Ca2+ responses required extracellular
Ca2+ (not shown).
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Inhibition of adenylyl cyclase
A membrane-permeant AC inhibitor was used to test whether OSNs needed the
ability to generate cAMP to produce odor-elicited Ca2+ responses.
As described below, cells that initially responded to one or more amino acid
were treated with MDL-12,330A (Guellaen
et al., 1977) to inhibit AC, then retested. Of 14 OSNs
tested in this way, inhibition of AC blocked the Ca2+ responses in
nine cells (64%), but was ineffective in five (36%), indicating that cAMP is
necessary for transducing some but not all odor responses.
An example of an OSN whose amino acid-induced Ca2+ response was blocked following inhibition of AC is shown in Figure 4. During initial testing, the cell responded to histidine and the response was mimicked by IBMX; a second cell tested simultaneously responded to IBMX only. Following incubation in MDL-12,330A, the responses to histidine and IBMX were eliminated in both cells, indicating their dependence on cAMP. Nevertheless, 8-br-cAMP was still able to elicit an increase in [Ca2+] in both cells, demonstrating that the elements of the AC/cAMP transduction pathway downstream of the cyclase remained functional.
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In some OSNs that responded to multiple amino acids, inhibition of AC by MDL-12,330A blocked some but not all responses. Two such cells that were recorded from simultaneously are shown in Figure 5. The initial chemosensitivity of these cells differed, with one cell responding to histidine, proline and taurine and the other responding to asparagine and taurine. Following treatment with MDL-12,330A, only the taurine responses remained. This suggests that the taurine responses were mediated by an intracellular pathway different from the AC/cAMP pathway.
Involvement of PLC
Inhibition of AC failed to block the Ca2+ responses elicited by one or more amino acid in 5 of the 14 cells tested. To determine whether odor responses that were insensitive to AC inhibition might be sensitive to inhibition of the PLC pathway, the PLC inhibitor neomycin was used. Neomycin (1 mM) was dissolved in APS and co-applied at neutral pH with amino acids. Figure 5B shows two cells whose responses to taurine persisted after treatment with MDL-12,330A to inhibit AC. The taurine responses in both cells were reversibly blocked by neomycin. This indicates that the taurine responses in these cells were not transduced by the AC/cAMP pathway and it suggests that the responses may have been transduced by the PLC pathway.
Thirty OSNs gave useful data with neomycin. By itself, neomycin caused no Ca2+ responses (not shown), but it blocked Ca2+ responses that were regarded as cAMP-independent. Most of the cAMP-independent responses were elicited by taurine; in six of seven taurine-responsive cells, taurine responses were unaffected by the AC inhibitor MDL-12,330A, while one was blocked. In contrast, the taurine responses in 14 of 15 taurine-responsive OSNs were blocked by neomycin; this included six cells pretreated with MDL-12,330A and eight cells not pretreated; the latter group indicate that neomycin's effect was independent of AC inhibition. Neomycin also blocked a response to leucine in one cell. To demonstrate that the blocking action of neomycin was not a non-specific effect, it was tested on cAMP-dependent responses elicited by IBMX and amino acids in cells that had not been treated with MDL-12,330A. Neomycin failed to block the responses to IBMX in 11 cells; in three other cells that individually responded to histidine, arginine or asparagine, the responses were both insensitive to neomycin and mimicked by IBMX or IBMX plus the forskolin analog L-858051 (see below). Taken together, these data suggest that neomycin selectively blocked cAMP-independent responses and failed to block responses mediated by the cAMP/AC pathway. These effects of neomycin are consistent with the idea that some but not all of the amino-acid-induced responses were transduced using the PLC pathway.
Interactions between the AC/cAMP and PLC pathways
Of the three cells noted above in which neomycin failed to block the amino acid responses, the responses in two cells appeared to be potentiated by neomycin. An example is shown in Figure 6; not only was the histidine response in Figure 6A potentiated by neomycin, but it was mimicked by IBMX plus 30 µM forskolin analog L-858051, suggesting that while the response to histidine was transduced by the AC/cAMP pathway, it was modulated by the PLC pathway. To study modulation of the AC/cAMP pathway by PLC, we examined how neomycin affected Ca2+ responses elicited directly by IBMX. The use of IBMX preserved the AC/ cAMP pathway intact, but eliminated the need to find amino acids that stimulated it. An example of potentiation of the response to IBMX by neomycin is shown in Figure 6B. Of 11 OSNs studied in this way, neomycin potentiated the IBMX response in eight cells by 2-10-fold and caused a partial inhibition of the response in one cell. These results suggest that the PLC pathway can modulate the AC/cAMP pathway, frequently but not exclusively by suppression. Thus the PLC pathway may serve roles in both the transduction of odor responses and their modulation.
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We examined whether the reciprocal form of modulation might also occur by comparing the magnitudes of MDL-12,330A-resistant, amino-acid-induced responses measured before and after treatment with the AC inhibitor. The data were from the five OSNs discussed earlier. Although the responses following treatment with MDL-12,330A were consistently smaller than before treatment, the changes were similar to normal rundown. Thus the data provided no convincing evidence that responses mediated by the PLC pathway were modulated by the AC/cAMP pathway; however, the data are insufficient to definitively rule out this possibility.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The importance of the AC/cAMP pathway as a mediator of vertebrate odor transduction is well established; however, the role of the PLC pathway in transduction has been less clear, especially for vertebrates. Some studies have suggested that the PLC pathway can mediate transduction, while others have suggested that it is a modulator of transduction but not a mediator (Zufall and Munger, 2001
).
This distinction is clear when modulation is caused by a stimulus such as a
hormone, but less obvious when both pathways are activated by odor. In this
study we have examined the issue using Necturus OSNs; the data
suggest that both the AC/cAMP and PLC pathways can act in the transduction of
odorants and that the PLC pathway also has a role in modulation.
Our approach was to measure changes in intracellular [Ca2+]
induced by odorants and use pharmacological tools to detect and distinguish
biochemical pathways. While action potentials, not changes in intracellular
[Ca2+], are the signals that communicate olfactory information to
neurons in the olfactory bulb, Ca2+ is a reliable indicator of
odor-induced receptor potentials in many if not all OSNs. The Ca2+
change begins in the cilia where odors stimulate ORs and it propagates to the
cell body as depolarization opens voltage-gated Ca2+ channels
(Leinders-Zufall et al.,
1997,
1998). Thus, while the
increase in [Ca2+] throughout the cell is clearly secondary, it
reflects the transduction events. The use of drugs to block or stimulate
selected pathways allowed us to monitor the differential effects of these
changes in individual OSNs, strengthening the evidence of two
pharmacologically distinct pathways.
Approximately two-thirds of the amino-acid-induced Ca2+ responses in Necturus OSNs were blocked by inhibition of the AC/cAMP pathway and a similar portion of responses were mimicked either by a cAMP analog or stimulation of AC. These results must be interpreted together, for mimicry alone, while consistent with transduction by the AC/cAMP pathway, cannot be distinguished from responses mediated by alternate pathways. The combined results support both the necessity and sufficiency of the AC/cAMP pathway in the transduction of the majority of olfactory responses. In addition, one-third of the odor responses were unaffected by AC inhibition or cAMP stimulation, suggesting that at least one other intracellular biochemical pathway is used for odor transduction. While these latter results might be expected if the concentrations of antagonists/agonists were too low to elicit effects in the unaffected cells, the observation of OSNs with heterogeneous responses makes this an unlikely explanation. OSNs with heterogeneous responses had one or more amino acid responses blocked by inhibition of AC, while the responses to other amino acids were unaffected. This provided evidence of the efficacy of the AC inhibitor, despite its inability to block all responses. The results from OSNs with heterogeneous responses not only support the main conclusion that multiple biochemical pathways are used for odor transduction, but also suggest that more than one transduction pathway operates in some OSNs. These data do not, however, allow us to determine whether multiple transduction pathways occur in all OSNs.
Our data suggest that some, if not all, of the amino acid responses not
mediated by the AC/cAMP pathway may be transduced through the PLC pathway.
Neomycin was an effective blocker of responses that were insensitive to
inhibition of the AC/cAMP pathway and neomycin is a potent blocker of PLC
(Striggow and Bohnensack,
1994). Taurine responses, in particular, were blocked by neomycin.
In agreement with earlier reports (Dubin
and Dionne, 1993), taurine was the most effective of the amino
acids tested in eliciting responses from Necturus OSNs (29% of cells,
Table 1). While it may be
tempting to think that the amino acids taurine and leucine, whose responses
were blocked by neomycin, might be transduced only through a PLC pathway, that
seems unlikely; both amino acids gave responses in other cells that were
insensitive to neomycin, blocked by inhibition of AC, or mimicked by cAMP
analogs. These odorants and possibly others may be transduced by activating
different ORs, one type linked to the AC/cAMP pathway, the other to the PLC
pathway. Alternatively, their receptors may couple to both pathways. This
latter possibility, however, seems inconsistent with data from several
taurine-responsive cells in which both pathways were present and mediated the
transduction of different amino acids, but neomycin and MDL-12,330A appeared
to affect separate amino acid responses and were not additive. This apparent
diversity of odor response mechanisms could explain an earlier report
(Chen et al., 2000)
that blocking AC in dissociated salamander OSNs inhibited responses to odors
that were assumed to activate only the PLC pathway.
Besides being a non-specific PLC inhibitor, neomycin can also block some
types of Ca2+ channels, especially in mammalian muscle and brain
preparations (Perrier et al.,
1992; Langton et al.,
1996). If neomycin blocked voltage-dependent Ca2+
channels in Necturus OSNs, that could explain the disruption of
odor-elicited Ca2+ responses that we observed. However, several
observations suggest that the action of neomycin in these experiments was not
produced by channel block. First, neomycin inhibited only a subset of the
Ca2+ responses elicited by amino acids. If neomycin were blocking
Ca2+ channels, it should inhibit all odorant-elicited responses.
Second, rather than inhibiting some Ca2+ responses, neomycin
potentiated them. This is inconsistent with channel block, but could be
explained if inhibition of PLC relieved suppression of the AC/cAMP pathway.
Finally, Ca2+ is reported to compete with neomycin in the block of
Ca2+ channels (Parsons et
al., 1992; Langton et
al., 1996); block is rapidly reduced as the [Ca2+]
is increased above 1 mM. In the experiments described here, we used 8 mM
Ca2+, a concentration that effectively minimizes neomycin block in
the preparations where it occurs. It should also be noted that the mechanism
of Ca2+ channel block by neomycin is unclear. Most of the reports
on this subject are consistent with either a direct action in which neomycin
might occlude the channel pore, or an indirect action mediated by inhibition
of PLC.
It has been demonstrated (Frings,
1993) that factors affecting protein kinase C, one of the enzymes
regulated by the PLC pathway, can enhance or attenuate the AC/cAMP pathway by
affecting AC performance. More recently, Gomez et al. (Gomez et
al.,
2000a,b)
reported modulation of odor-induced Ca2+ responses in rat and human
OSNs by protein kinases, where an inhibitor of protein kinase C potentiated
AC/cAMP-mediated odor responses and an inhibitor of cAMP-dependent protein
kinase A potentiated PLC-mediated responses. Our data suggest that the PLC
pathway can modulate responses transduced by AC/cAMP in Necturus
OSNs, similar to the results of Gomez et al., but we saw no evidence
that the AC/cAMP pathway modulated PLC-mediated responses elicited by amino
acids in these cells. Inhibition of the PLC pathway led to larger
amino-acid-elicited Ca2+ responses in several cells. When this
effect was examined using IBMX to stimulate responses, it appeared that the
Ca2+ responses in most OSNs were potentiated after PLC was
inhibited, suggesting that this form of modulation is widespread if not
ubiquitous. Interactions between these pathways may underlie the non-monotonic
dose—response relations seen using IBMX as a stimulus. Modulation of
olfactory responses in vivo may be produced by neuroactive compounds
such as hormones or neuropeptides. Nevertheless, the observations that the PLC
pathway may also be involved in the transduction of certain odorants and that
transduction events mediated by this and the AC/cAMP pathway can be observed
in the same OSNs, suggest that complex interactions between the pathways
during odor transduction may occur in some cells. In these cells, stimulation
of the PLC pathway by an odorant could modulate transduction events mediated
by the AC/cAMP pathway. The consequences would affect odor perception if the
affected cells were sensitive to certain stimuli, for then the sensitivity of
the olfactory system to particular odors could be selectively enhanced or
suppressed.
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Acknowledgments |
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We would like to thank Drs Sue Kinnamon, Diego Restrepo and Tatsuya Ogura for their generosity and advice—without it, this study might never have been undertaken and it certainly would not have been completed. This work was supported by grants from the National Institutes of Health—DC03912 and RR16435 (R.J.D.) and DC00256 (V.E.D.).
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Accepted June 13, 2002
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