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Proc Natl Acad Sci U S A. Aug 2, 2005; 102(31): 11100–11105.
Published online Jul 22, 2005. doi:  10.1073/pnas.0501988102
PMCID: PMC1182420
Neuroscience

Expression, physiological action, and coexpression patterns of neuropeptide Y in rat taste-bud cells

Abstract

Recent studies have suggested that neuropeptides could play previously unrecognized functional roles in peripheral gustation. To date, two peptides, cholecystokinin and vasoactive intestinal peptide, have been localized to subsets of taste-bud (TB) cells (TBC) and one, cholecystokinin, has been demonstrated to produce excitatory physiological actions. This study extends our knowledge of neuropeptides in TBC in three significant ways. First, using techniques of immunocytochemistry and RT-PCR, evidence is presented for the expression of a third peptide, neuropeptide Y (NPY). Like other peptide expression patterns, NPY expression is circumscribed to a subset of cells within the taste bud. Second, using physiological studies, we demonstrate that NPY specifically enhances an inwardly rectifying potassium current via NPY-Y1 receptors. This action is antagonistic to the previously demonstrated inhibitory effect exerted by cholecystokinin on the same current, thus providing important clues to their signaling roles in the TB. Third, using the technique of double-labeled fluorescent immunocytochemistry, the relationship of three subsets of neuropeptide-expressing TB cells to one another was examined. Remarkably, NPY expressions, although fewer in number than either the cholecystokinin or vasoactive intestinal peptide subsets, overlapped 100% with either peptide. Collectively, these three observations transform previously suggestive roles of neuromodulation by peptides in TB cells to more concrete signaling pathways. The extensive colocalization of these peptides suggests they may be subject to similar presynaptic influences of release yet have antagonistic postsynaptic actions. The convergence or divergence of these postsynaptic actions awaits further investigation.

Keywords: gustation, neuromodulation, sensory transduction

Mechanisms that taste-bud (TB) cells (TBCs) use to signal the central nervous system of the presence of gustatory stimuli in the oral cavity remain incompletely understood. These mechanisms include both early events in single cells, i.e., receptor-mediated stimulation that initiates changes in cellular excitability, and late events among cells, intercellular signaling with neurotransmitters and neuropeptides before peripheral afferent nerve activation. Although advances in molecular biology have yielded new evidence on early mechanisms, late events have been more appreciated only recently. These late events may rely strongly on the tightly cloistered arrangement of TBCs in the TB, long understood to be a universal feature of vertebrate gustation, although the physiological advantages of this highly conserved morphology are not well understood.

TBCs are differentiated epithelial cells clustered into unique morphological arrangements termed buds that place 50-100 individual cells into close apposition to one another. Only a minority of these cells are synaptically connected to the central nervous system via sensory afferent nerve fibers (e.g., ref. 1). It is believed that this specialized morphology facilitates cell-to-cell communication among TBCs. There is now evidence for multiple neurotransmitters and neuropeptides within the mammalian TB. These include transmitters such as glutamate (2, 3), norepinephrine (4, 5), serotonin (6-9), acetylcholine (10, 11), and possibly GABA (12), as well as two neuropeptides, cholecystokinin (13) and vasoactive intestinal polypeptide (14, 15).

Of these signaling agents, neuropeptides are the most recently discovered and hence the least understood. Both cholecystokinin (CCK) and vasoactive intestinal peptide (VIP) are members of the brain-gut family of peptides, are widely distributed in the central and peripheral nervous systems, and function as neurotransmitters and neuromodulators. It has been proposed that these peptides play similar roles within the TB. CCK has been demonstrated to alter electrical activity of TBCs and to elevate intracellular calcium levels (13), and it is correlated to bitter responsiveness (10). Both CCK and VIP have overlapping expression patterns with gustducin (GUST) and T1R2, suggesting that they could operate in transduction mechanisms (16). Additionally, CCK acts to modulate responses in the chorda tympani nerve (17).

This manuscript extends our knowledge of neuropeptides within the TB by presenting three observations. First, we report evidence for the expression of a third neuropeptide in TBCs, neuropeptide Y (NPY), also a member of the brain-gut peptide family. Second, physiological experiments demonstrated that NPY exerts inhibitory actions on single TBCs that are opposite in nature to those exerted by CCK. Third, NPY-expressing cells form a completely overlapping subset of CCK- and VIP-expressing TBCs. Collectively, these data not only establish NPY as a recently discovered neuromodulator in TBs but additionally provide strong evidence to firmly establish neuropeptides in general as signaling agents that likely act in concert with transduction mechanisms to produce the final afferent signal that is relayed to the central nervous system.

Experimental Procedures

Animals. Adult male Sprague-Dawley rats were anesthetized with 0.09 ml/100 gm of body weight of a 91-mg/ml ketamine/0.09-mg/ml acepromazine mixture, killed, and gustatory papillae were quickly dissected. All procedures were approved by Ohio State University's Laboratory Animal Care and Use Committee and adhered to the National Institutes of Health “Guide for the Care and Use of Laboratory Animals.”

Immunocytochemistry. Paraffin-embedded Bouin's fixed papillae were sectioned at 8-μm thickness and processed with standard immunocytochemical protocol by using a primary antiserum against a neuropeptide (NPY, DiaSorin; Stillwater, MN; CCK, Chemicon; VIP, DiaSorin) at dilutions ranging from 1:1,000 to 1:20,000, respectively, a secondary biotinylated goat-anti-rabbit IgG diluted to 1:400 in PBS-TX, and an avidin-biotin peroxidase complex (Vectastain “Elite ABC” kit, Vector Laboratories) reacted with 3-3′-diaminobenzidine tetrahydrochloride (in 0.05 M Tris buffer, pH 7.6) and hydrogen peroxide. Omission of either the primary or secondary antibody eliminated all staining.

Because commercially available antibodies for the peptides of interest are all raised in rabbit, double-labeling immunocytochemical experiments were performed with a modified indirect immunofluorescence protocol that allowed localization of two antigens in the same preparation with both primary antibodies from the same species. This protocol utilizes tryamine signal amplification (TSA; NEN Life Science) for detection of the first primary antibody, which is run at very high dilution so that it is not observed with standard detection methods. Additionally, it is visualized with a Fab fragment secondary antibody to prevent crossreaction between the first primary antibody and the second secondary antibody (see ref. 18 for details).

Two experiments were performed to make certain that unwanted crossreactivity did not occur. Each controlled for one of the two possible patterns of crossreactivity (diagrammatically presented in Fig. 7, which is published as supporting information on the PNAS web site), either the second primary antibody crossreacting with the first secondary antibody (interference I) or the first primary antibody crossreacting with the second secondary antibody (interference II). In principle, the experimental design precludes the occurrence of interference I. Because a Fab fragment is used as the first secondary antibody, additional antibody-binding sites are lacking. Therefore, when the second primary is applied, there should be no binding sites available for the unwanted capture of the second primary by the first secondary (interference I). The lack of such crossreactivity was verified in control experiments that used a second primary antibody known not to be expressed in lingual tissue, ionized calcium-binding adaptor molecule 1 (Iba1), a microglia/macrophage-specific calcium-binding protein. Because lingual tissue lacks the Iba1 antigen, its visualization would be detected only if crossreactivity were occurring. Tissue was first incubated with a CCK primary antibody and a Fab fragment FITC-conjugated secondary and TSA amplification. Immunopositive cells were observed (Fig. 7B). Tissue was subsequently incubated with antibody directed against Iba-1 (1:100 dilution) and visualized, using standard methods, with a Cy3-conjugated secondary antibody. Parallel experiments with brain tissue at this dilution produced specific labeling in microglia (not shown). Results of these experiments demonstrated complete lack of any cellular staining with Iba1 antibody, demonstrating that the unwanted capture by the goat-anti-rabbit FITC-conjugated antibody was prevented because the Fab fragment lacks an available binding site for the Iba-1 antibody (Fig. 7C).

To control for interference II, visualization of the first primary antibody at the working dilution for amplified detection was tested by using standard detection methods rather than TSA. Because the first primary antibody is applied at very high dilution, only the amplified means of detection should be able to visualize its binding. To verify this prediction, in control experiments, detection of each primary antibody was tested at its working dilution for TSA amplification and tested with standard visualization. For example, detection of VIP-positive TBCs using a dilution 10 times higher than for conventional immunofluorescence is presented by using the TSA protocol with FITC detection (Fig. 7D). When VIP primary antibody was applied followed by serum replacement for the first secondary antibody, and the second secondary reacted with standard (i.e., nonamplified) Cy3 detection, no signal could be detected (Fig. 7E). This demonstrates detection of VIP antibody at this dilution is possible only with amplified means, and hence crossreactivity representative of interference II is not occurring. Similar results were obtained when using CCK as the first primary antibody.

RT-PCR. RT-PCR experiments were performed on total RNA isolated from individually harvested circumvallate TBs (20-50) collected into a 1.5-ml microtube containing 100 μl of TRIzol reagent. RNA was treated for 30 min with amplification grade DNase-I. First-strand cDNA was synthesized from total RNA extracted from pure TBs or brain using oligo(dT)12-18 primer. Subsequently, the following components were added to the reaction, with a final total volume of 20 μl: 1× of first-strand buffer/10 mM DTT/500 μM each dNTP/200 units of SuperScript II RNase H-Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA).

A previously published (19) primer set specific for the rat NPY gene was used: forward primer, 5′-GCT AGG TAA CAA ACG AAT GGG G-3′; reverse primer, 5′-CAC ATG GAA GGG TCT TCA AGC-3′. PCR was carried out in a volume of 50 μl by using 1 μl of cDNAs for each reaction. The standard reaction mixture consisted of 1× PCR buffer (200 mM Tris·HCl, pH 8.4/500 mM KCl)/0.2 μM forward and reverse primer/0.2 mM each dNTP/1.5 mM MgCl2/2.5 units of Platinum TaqDNA polymerase (Invitrogen Life Technologies). The PCR profile was 94°C at 5 min (one cycle), 94°C at 30 sec, 55°C at 30 sec, 72°C at 45 sec (35 cycles), and 72°C at 10 min (one cycle). Additionally, in early experiments using RNA extracted from lingual epithelium (as opposed to pure TBs), a one-tube RT-PCR strategy with the same gene-specific primers using SuperScript One-Step RT-PCR with Platinum Taq Kit (Invitrogen Life Technologies) according to manufacturer's instructions was also used and served as a different approach to confirm our results.

Controls for DNA contamination and PCR carryover were performed. These included omission of the reverse transcriptase enzyme (RT-) to control for genomic contamination and omission of template (H2O) control, respectively. Cerebral cortex was dissected and RNA extracted and run in parallel with experimental reactions as a positive control tissue. Additionally, reactions using primers for a housekeeping gene (β-actin) and for a robustly expressed taste-specific gene (GUST) were performed (β-actin, 5′-CAC GGC ATT GTA ACC AAC TG-3′; reverse primer, 5′-TAA TGT CAC GCA CGA TTT CC-3′; GUST, 5′-GTT GGC TGA AAT AAT TAA ACG-3′; reverse primer, 5′-ATC TCT GGC CAC CTA CAT C-3′). PCR products were visualized by using 1.5% agarose gel stained with ethidium bromide. PCR products were purified by using the Concert Rapid PCR Purification System (Invitrogen Life Technologies). Purified samples and primers were submitted to the Plant-Microbe Genomic Facility at the Ohio State University facility for sequencing.

Physiological Analysis. Patch-clamp experiments were performed on TBCs dissociated from circumvallate and foliate papillae, as described (e.g., refs. 6-13). Data were acquired with a high-impedance amplifier and a commercial software program (axopatch 1b; pclamp; Axon Instruments, Union City, CA). Membrane currents were acquired after low-pass filtering with a cutoff frequency of 10 kHz (at -3 dB). Inwardly rectifying potassium current (Kir) was isolated as reported (20). The membrane voltage was typically held at its zero-current potential (in 100 mM extracellular potassium), usually around -3to -10 mV, and a series of depolarizing or hyperpolarizing command potentials, in 10-mV increments, was applied ranging from -160 mV to +30 mV (20). Leak subtraction was not used. Data were analyzed with a combination of off-line software programs that included a software acquisition suite (pclamp) and a technical graphics/analysis program (origin 6.0; MicroCal Software, Northampton, MA). Exchange of the bathing solution was accomplished with a gravity-fed perfusion system. Data were normalized to the value of the current magnitude before drug application. Fisher's exact probability test was used to evaluate statistical difference between means, considered significant at values of P < 0.05. Data are presented as mean ± SE.

Intracellular calcium levels in dissociated TBCs were monitored by using standard ratiometric techniques with the calcium-sensitive dye fura-2 (Molecular Probes) and a commercially available software package for data acquisition and analysis (simplepci, Compix, Cranberry Township, PA) as described (10, 13).

Results

Distribution of NPY-Like Immunoreactivity in TBC of the Oral Cavity. TBCs containing reaction product for NPY-like immunoreactivity were observed in TBs of the nasoincisor ducts and the foliate, circumvallate, and fungiform papillae by using a commercially available primary antibody to NPY visualized with standard colorimetric techniques (Fig. 1). Positive cells displayed a characteristic distribution of reaction product that was confined to the cytosol with clear nuclei. Reaction product extended evenly throughout the cytoplasm from the apical to the basal end of the cell without evidence of polar distribution. No obvious difference was noted in the location of positive cells within a TB; positive cells were observed within the center or periphery of the bud. Rat-brain cortex was included as a positive control tissue for NPY immunoreactivity (not illustrated). Numerous immunopositive neurons were observed in rat cortex, where NPY-expressing cells are abundant in layers II and III. Omission of the primary antibody or secondary antibody eliminated all staining.

Fig. 1.
Photomicrographs of TBs containing NPY-immunopositive TB cells from circumvallate (CV), foliate (FOL), and fungiform papillae (AT) of the rat tongue and from the nasoincisor ducts (NID). (Bars, 20 microns.)

Localization of NPY Messenger RNA to TBs by RT-PCR. To verify the expression of NPY in TBCs, RT-PCR experiments on RNA isolated from individually collected circumvallate TBs were conducted. Single buds were pooled, lysed, and total RNA was extracted. A previously published (19) NPY primer set was used (expected product size, 288 bp). RT-PCR experiments included positive control primer sets for GUST (expected product size, 231 bp), a G protein highly expressed in many TBCs, and β-actin (not shown) to verify the integrity of the extracted RNA. RT-PCR conditions for the NPY primer set were optimized on RNA extracted from cerebral cortex, which was used as a positive control tissue. All experiments were performed with parallel negative control experiments that either omitted the reverse transcriptase enzyme (RT-) or template (H2O control) and yielded the expected results. PCR product derived from TB template using NPY primers was sequenced to confirm its identity. Results are illustrated in Fig. 2. Bands of expected size for positive control tissues, GUST in TBs, and NPY mRNA in rat cortex, were observed. Bands for PCR products indicative of NPY mRNA were observed in two experimental samples, pure TB as well as lingual epithelium containing TBs. PCR with (+) or without (-) inclusion of reverse transcriptase is illustrated for each primer set. These data verify NPY expression in TBCs by confirming the presence of its mRNA.

Fig. 2.
RT-PCR detection of rat NPY mRNA in posterior TBs. Electrophoresis of PCR products revealed bands with expected sizes for NPY mRNA (288 bp) in total RNA isolated from TBs obtained from circumvallate and foliate papillae (TBs), lingual epithelium containing ...

Physiological Actions of NPY on TBC. Physiological actions of exogenously applied NPY on dissociated TBCs were examined in experiments using either patch-clamp analysis of macroscopic currents or ratiometric imaging of intracellular calcium. In both sets of experiments, NPY peptide was applied focally to dissociated TBCs with a stimulating pipette positioned close to the cell against a background flow of extracellular fluid. Using patch-clamp analysis, macroscopic sodium and outward potassium currents were observed to be unaffected by exogenous NPY application at concentrations up to 1 μM. However, when isolated inwardly rectifying potassium currents were tested (previously characterized in ref. 20), the current magnitude was significantly enhanced in a subset of TBCs (Fig. 3 Upper). This enhancement occurred over the inwardly rectifying portion of the current and was reversible with a rinse of the peptide from the bathing solution. NPY concentrations of 1, 10, 100, or 500 nM were tested and produced maximal enhancements of the current of 117 ± 1.7% (3 of 18 cells tested, or 16%), 123 ± 3.2% (16 of 42 cells, 38%), 135 ± 9.5% (14 of 35 cells, 40%), or 124 ± 4.4% (8 of 21 cells, 38%), respectively (Fig. 3 Lower). The percentage of responding cells did not significantly vary with NPY concentration. Cells that did not respond to NPY, when pooled, produced no response (101 ± 1.1%, n = 66). These observations suggest that NPY responses likely depend upon the expression or absence of NPY receptors.

Fig. 3.
Application of exogenous NPY to dissociated TB cells enhances Kir. (Upper) Sample current traces of Kir, obtained with whole-cell patch-clamp recording, are presented from a single TB cell before ([multiply sign in circle]), during ([down-pointing small open triangle]), and after ([plus sign in circle]) ...

To test for receptor mediation of this response, both NPY receptor agonist and antagonist were used. BIBP3226 [(R)-N 2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-d-argininamide], an NPY-1 receptor antagonist, was observed to significantly decrease the response produced by either 10 or 100 nM NPY (Fig. 4A; P < 0.005). Separate populations of TBCs were probed for either NPY responsiveness or NPY responsiveness after previous application of 1 μM BIBP3226 to make certain that receptor desensitization did not produce false-negative results. At either NPY concentration, ≈35-40% of TBCs responded, which was reduced to 7% after BIBP3226 application. To further explore the NPY receptor subtype, the NPY-1 receptor agonist [Leu-31, Pro-34]-NPY was tested. At 10 nM, 7 of 21 cells tested responded (33%), and at 100 nM, 13 of 36 cells tested responded (36%; Fig. 4B). However, if either concentration was preceded by exposure to 1 μM BIBP3226, only 7% or 8% (10 and 100 nM, P < 0.03, P < 0.02, respectively) responded. Thus, these data strongly imply NPY responses to be mediated by the NPY-1 receptor subtype.

Fig. 4.
NPY actions on Kir may be mediated by NPY-1 receptors and are G protein-dependent. (A) Approximately 40% of all tested cells responded to either of two tested concentrations of NPY (10 and 100 nM). However, if cells were treated with the NPY-1 receptor ...

To begin an initial characterization of the underlying signal transduction mechanism, G protein involvement and possible signaling enzymes mediating the Kir enhancement were pharmacologically explored. G protein involvement was tested with the inclusion of 2 mM GDP-βS, a nonhydrolyzable GTP analogue that inactivates G proteins by irreversibly binding to the alpha subunit, to the patch pipette in whole-cell recording configuration. Kir has previously been demonstrated to be G protein-dependent in TBCs (20). Although 38% of tested cells responded to 10 nM NPY using standard intracellular fluid (ICF), only 9% (3/32 cells) responded with GDP-βS ICF (Fig. 4C). This effect achieved statistical significance (P < 0.005) and suggests NPY-1 receptors use G proteins in enhancing Kir.

In an attempt to address the mechanism of this enhancement, two treatments were performed. First, the effect of the specific phosphatidylinositol 3-kinase blocker, LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] was tested [50 μM added to intracellular fluid (ICF)] on the response to 10 nM NPY. Seven of 23 tested cells (30%) responded (Fig. 4C). Second, the possible involvement of phospholipase C was tested by using its specific inhibitor U73122 [{1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione}]; 125 μM added to the ICF). Ten of 27 cells (37%) tested with 10 nM NPY responded. Neither treatment approached statistical significance when compared with the NPY response alone. These results suggest neither enzyme is involved but instead the Kir channel is activated by a direct interaction of the βγ subunit of the G protein.

In contrast to results observed with patch-clamp analysis, effects of NPY application on intracellular calcium levels, tested with calcium imaging, were not evident. Exogenous NPY (0.5 μM) was focally applied to dissociated TBCs by using ratiometric imaging with the calcium indicator fura-2. Thirty-one cells were tested; 28 cells gave no response to NPY, as indicated by a lack of change of ratio baseline during peptide administration. Three cells gave possible indications of response (9%). All cells were tested with other agents known as effective stimuli for these cells (20 mM caffeine and/or 10 μM acetylcholine), which served as positive controls. It was concluded that an obvious connection between NPY administration and calcium elevations could not be demonstrated.

Colocalization Patterns of NPY with Other Peptides. To examine the colocalization pattern of NPY with the peptides CCK or VIP, double-labeling immunocytochemistry was performed on rat posterior TBs by using rabbit primary antibodies directed against these peptides and the fluoroprobes FITC or CY3. The staining pattern for all three antigens of interest in the double-labeling fluorescent protocol was qualitatively similar to that observed with the single-labeling protocol.

Results of double-labeling experiments with antibodies against NPY and CCK demonstrated two patterns of immunopositive cells. Most displayed immunoreactivity for both antigens, whereas a smaller subset of labeled cells displayed immunoreactivity for CCK alone. Single-labeled NPY cells were rarely observed. Examples of the nearly complete coincident labeling of NPY with CCK from rat circumvallate papillae are presented in Fig. 5 A-C. Photomicrographs in the left column (Fig. 5 A, D, and G) are single-wavelength unamplified detection using Cy3 fluorochrome, in the center column (Fig. 5 B, E, and F), the single-wavelength amplified detection using FITC; and the right column (Fig. 5 C, F, and I) presents the overlay of the two single-wavelength excitation images. In overlay images, double-labeled cells appear yellow. The scale bar represents 20 microns and applies to all images. A total of 371 TBs from three separate experiments were examined for quantitative analysis. Within these TBs, 34 TBCs were positive for NPY only, 288 TBCs were positive for CCK only, and 597 taste cells showed colocalization of both antigens. The double-labeled TBCs represent ≈68% of all CCK-containing cells and 95% of all NPY-containing cells (Table 1).

Fig. 5.
NPY-expressing TBC colocalize completely with CCK- or VIP-expressing TBC. Representative photomicrographs of double-labeling immunocytochemical experiments performed with the fluorochromes FITC (green) and CY-3 (red) on rat posterior TBs are presented. ...
Table 1.
Quantitative analysis of colocalization of NPY, CCK, and VIP in rat posterior TBCs

In the next combination of double-labeling experiments, colocalization of the neuropeptides NPY and VIP was compared. Results of the NPY/VIP combination were strikingly similar to those produced in the NPY/CCK experiments. Two major types of labeled subsets of TBCs emerged from these experiments. Most labeled cells demonstrated fluorescence to both fluoroprobes demonstrating double label for both NPY and VIP, whereas fewer labeled TBCs were immunopositive for VIP alone. Cells were rarely observed that labeled for NPY without coincident VIP labeling. Representative photomicrographs from foliate tissue are illustrated in Fig. 5 D-F. Essentially complete colocalization was again observed for NPY-expressing TBCs. A total of 378 TBs from three separate experiments were evaluated. Of these TBs, only 14 TBCs were positive for NPY alone, 399 were positive for VIP alone, and 573 showed colocalization of both antigens. The double-labeled TBCs represent ≈59% of all of VIP-expressing cells and 98% of all of the NPY-expressing cells (Table 1).

To complete the examination of coexpression patterns, a final series of experiments with double-labeling immunocytochemistry was performed by using two primary antibodies directed against CCK or VIP. With this combination, the majority of cells displayed immunofluorescence to both peptides. Additionally, single-labeled TBCs, although fewer in number, for either CCK or VIP were also observed. In three separate experiments, the majority of labeled TBCs displayed both Cy3 and FITC emission. Sample data from rat foliate TBs are presented in Fig. 5 G-I. A total of 307 TBs were evaluated. Of these TBs, 168 were positive for CCK only, 197 were positive for VIP alone, and 527 demonstrated colocalization of both antigens. These double-labeled cells represent 76% of all of CCK-expressing cells and 73% of all of the VIP-expressing cells (Table 1). A quantitative illustration of the overlapping distribution pattern of the expression of these neuropeptides is presented in Fig. 6.

Fig. 6.
A quantitative illustration of the overlapping distribution pattern of the expression of the neuropeptides CCK, VIP, and NPY in rat TBC. Each square represents the size of the subset of peptide-expressing cell observed, normalized to the CCK distribution. ...

Discussion

The report of expression of NPY in a subset of rat TBCs and of a significant physiological response to its exogenous application significantly extends the role of neuropeptides as signaling agents in TBs. These observations are of parallel significance to those reported (13) for another neuropeptide, cholecystokinin. Both demonstrate peptide expression in subsets of TBCs and physiological actions mediated specifically by neuropeptide receptors. Together, these previously unknown phenomena lend strong credence to the hypothesis that peptides play important roles in information processing among the cells of the TB. Specific autocrine/paracrine expression patterns across the TB of peptide-expressing TBCs and peptide-receptor-expressing TBCs delineate a hard-wiring pattern defining potential modulatory routes. That the physiological actions of CCK and NPY are antagonistic imparts more consequence to their signaling roles.

In rat TBCs, three complementary excitatory physiological actions of CCK were observed. These include inhibition of outward potassium currents, elevation of intracellular calcium from intracellular stores, and inhibition of Kir. All would serve to place the cell in a more excitatory state, in agreement with the notion of its fulfilling a neuromodulatory role. The strong desensitization of these responses predicts these physiological effects would be transient. Both effects are mediated by CCK-A receptor subtype. NPY, on the other hand, was observed to enhance Kir. As in many cells, this current contributes strongly to the resting potential of TBCs (20). Our observations suggest this to be mediated by the NPY-1 receptor subtype. Hence enhancement of this current would act to stabilize the resting potential, causing it to be less excitable. CCK, on the other hand, inhibits this current via CCK-A receptors. Given the apparent antithetical physiological actions of NPY and CCK, one observation of immediate importance is the relationship of the expression patterns of these two peptides to one another.

A major finding of the present studies is the remarkable colocalization patterns of these three peptides in single TBCs (Figs. (Figs.55 and and6,6, Table 1). Our working hypothesis of peptide expression in TBCs, presented quantitatively in Fig. 6, assumes complete overlap of the expression pattern of NPY with CCK and VIP. This hypothesis assumes the empirical value of NPY coexpression with these two neuropeptides (95% or 97%) is within experimental error of the double-labeling expression protocol and that, because CCK and VIP extensively coexpress with one another, TBCs expressing NPY also express both CCK and VIP. This large overlapping coexpression pattern implies some presynaptic functional commonality. For example, similar tastant stimuli may be linked via their transduction pathways to the release of these peptides. Two possibilities of convergent or divergent postsynaptic routes remain possible. Whereas CCK appears to operate in the TB in an autocrine manner (unpublished observations), the relationship of NPY- and NPY-1-expressing TBCs remains unknown. Hence, these two peptides may act simultaneously to excite and inhibit different sets of TBCs within the bud or could act sequentially to excite then inhibit the same sets of TBCs. Nevertheless, the unique expression pattern combined with the opposing physiological actions of two (CCK and NPY) lends further credence to the notion that neuropeptides play significant, although at present incompletely understood, roles in peripheral TB physiology.

One clue into the role(s) that neuropeptides play in the processing of peripheral taste information may come from the phenotyping subsets of TBCs that express either neuropeptide or neuropeptide receptor. A previous study that investigated the colocalization patterns of CCK and VIP with transduction molecules T1R2 and GUST (16) demonstrated that the majority of the neuropeptide-expressing TBCs coexpressed GUST and were infrequently colocalized with T1R2-expressing TBCs. Because the present results demonstrated NPY to be colocalized with CCK and VIP, it is predicted that NPY-expressing TBCs would display similar coexpression patterns. These data suggest that these neuropeptides are less likely to be involved in sweet transduction pathways, because they poorly colocalize with sweet receptors. One the other hand, their colocalization with GUST, which often coexpresses members of the T2R family of receptors, suggests involvement with bitter transduction to be more likely. It should be emphasized that, at present, these notions remain entirely inferential. Nevertheless, a recent study demonstrated that CCK-expressing TBCs responded well to the bitter stimuli quinine and caffeine (10).

Finally, it will be interesting to investigate whether neuropeptide-expressing TBCs are colocalized in a meaningful manner with conventional transmitters such as GABA or NE, both of which have been detected immunocytochemically in rat taste cells (5, 12). Neuropeptides are typically colocalized with classic neurotransmitters in other cell types. For example, in the central nervous system, NPY, CCK, and VIP coexist with GABA (21-23), and NPY is well known to be coexpressed with NE in sympathetic neurons (24, 25.). If this is true in TBCs, then the presence of more than one peptide in a single cell combined with the possibility of classical neurotransmitter may permit taste cells to transmit multiple messages upon taste stimulation.

Supplementary Material

Supporting Figure:

Acknowledgments

This work was supported by National Institutes of Health Grant DC00401 (to S.H.).

Notes

Author contributions: F.-l.Z., T.S., N.K., S.-g.L., Y.C., and S.H. designed research; F.-l.Z., T.S., N.K., S.-g.L., and Y.C. performed research; F.-l.Z., T.S., N.K., S.-g.L., Y.C., and S.H. analyzed data; and S.H. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: NPY, neuropeptide Y; TB, taste bud; TBC, TB cell; CCK, cholecystokinin; VIP, vasoactive intestinal peptide; TSA, tryamine signal amplification; GUST, gustducin; Kir, inwardly rectifying potassium current.

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