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RECEPTORS AND SIGNAL TRANSDUCTION

Bitter stimuli induce Ca²⁺ signaling and CCK release in enteroendocrine STC-1 cells: role of L-type voltage-sensitive Ca²⁺ channels

Division of Digestive Diseases, Department of Medicine, CURE: Digestive Diseases Research Center and Molecular Biology Institute, David Geffen School of Medicine, University of California at Los Angeles

Submitted 5 January 2006 ; accepted in final form 9 May 2006

	ABSTRACT

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We previously demonstrated the expression of bitter taste receptors of the type 2 family (T2R) and the -subunits of the G protein gustducin (G_gust) in the rodent gastrointestinal (GI) tract and in GI endocrine cells. In this study, we characterized mechanisms of Ca²⁺ fluxes induced by two distinct T2R ligands: denatonium benzoate (DB) and phenylthiocarbamide (PTC), in mouse enteroendocrine cell line STC-1. Both DB and PTC induced a marked increase in intracellular [Ca²⁺] ([Ca²⁺]_i) in a dose- and time-dependent manner. Chelating extracellular Ca²⁺ with EGTA blocked the increase in [Ca²⁺]_i induced by either DB or PTC but, in contrast, did not prevent the effect induced by bombesin. Thapsigargin blocked the transient increase in [Ca²⁺]_i induced by bombesin, but did not attenuate the [Ca²⁺]_i increase elicited by DB or PTC. These results indicate that Ca²⁺ influx mediates the increase in [Ca²⁺]_i induced by DB and PTC in STC-1 cells. Preincubation with the L-type voltage-sensitive Ca²⁺ channel (L-type VSCC) blockers nitrendipine or diltiazem for 30 min inhibited the increase in [Ca²⁺]_i elicited by DB or PTC. Furthermore, exposure to the L-type VSCCs opener BAY K 8644 potentiated the increase in [Ca²⁺]_i induced by DB and PTC. Stimulation with DB also induced a marked increase in the release of cholecystokinin from STC-1 cells, an effect also abrogated by prior exposure to EGTA or L-type VSCC blockers. Collectively, our results demonstrate that bitter tastants increase [Ca²⁺]_i and cholecystokinin release through Ca²⁺ influx mediated by the opening of L-type VSCCs in enteroendocrine STC-1 cells.

type 2 family taste receptors; gastrointestinal peptides; phospholipase C ₂; Ca²⁺ fluxes; enteroendocrine cells; cholecystokinin secretion

The gustatory system has been selected during evolution to detect nonvolatile nutritive and beneficial (sweet) compounds as well as potentially harmful (bitter) substances (24, 34). In particular, bitter taste has evolved as a central warning signal against the ingestion of potentially toxic substances, including plant alkaloids and other environmental toxins (21, 65). Specialized neuroepithelial taste receptor cells, organized within taste buds in human and rodent lingual epithelium, expressed a family of bitter taste receptors (referred as T2Rs) (1, 6, 46). These putative taste receptors belong to the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor (GPCR) superfamily (1), which are characterized by seven transmembrane -helices (32). Extensive genetic and biochemical evidence indicate that specific G proteins, gustducin and transducin, mediate bitter and sweet gustatory signals in the taste buds of the lingual epithelium (47, 48, 62, 63, 73). More recently, phospholipase C₂ (PLC₂) and TRPM5, a member (melastatin subtype 5) of the transient receptor potential (TRP) family (49), have been linked to bitter and sweet signal transduction (55, 56, 81). There is evidence for the activation of multiple second messenger pathways and ion channels in individual taste cells (1, 82). Clearly, taste signal transduction is complex and multifactorial and there is still much that is unknown about individual taste cell regulation.

Outside the tongue, expression of the -subunit of gustducin (G_gust) has been also localized to gastric (28, 75) and pancreatic (27) cells, suggesting that a taste-sensing mechanism may also exist in the digestive system. Indeed, we demonstrated the expression of members of the bitter taste receptors of the T2R family in the mouse and rat GI tract and in mouse and rat enteroendocrine cells in culture (74, 75). More recently, these results have been confirmed (45) and extended to the expression of the sweet taste receptors of the T1R family (15). Collectively, these findings demonstrated the expression of taste signal transduction pathways in cells of the GI tract of mice and rats.

The intracellular signal transduction cascades initiated by bitter stimuli in GI endocrine cells have not been explored. As a first step to examine these processes, we used the mouse enteroendocrine STC-1 cell line as a model system (61). These cells have been used for studying the regulation of GI hormone release in response to bombesin/gastrin-releasing peptide (10, 54, 67), pituitary adenylate cyclase-activating polypeptide (7), leptin (22), fatty acids (8, 26, 66), orexin (36), amino acids (10, 38, 41, 43, 67), and peptidomimetic compounds (50, 53). STC-1 cells produce and release cholecystokinin (CCK), glucose-dependent insulinotropic polypeptide, secretin, and glucagon-like peptide-1 (GLP-1) (8, 9, 11, 26, 38) and have also served as a model for studies of enteroendocrine cell differentiation (59). In the present study, we used STC-1 cells as a model system to elucidate Ca²⁺ signaling and secretory responses initiated by bitter tastants in enteroendocrine GI cells.

Activation of bitter taste receptors promotes the synthesis of second messengers leading to the release of Ca²⁺ from intracellular stores and/or modulates the gating of ion channels that mediate Ca²⁺ entry into neuroepithelial taste cell (25, 44). To determine whether these pathways also operate in enteroendocrine cells, we examined the effect of the structurally unrelated bitter stimuli denatonium benzoate (DB) and phenylthiocarbamide (PTC) on intracellular Ca²⁺ ([Ca²⁺]_i) in STC-1 cells. Our results demonstrate that these bitter tastants increase [Ca²⁺]_i through Ca²⁺ influx mediated by the opening of L-type voltage-sensitive Ca²⁺ channels (VSCCs) leading to CCK release in enteroendocrine STC-1 cells.

	MATERIALS AND METHODS

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Cell lines and DNA. The STC-1 cell line is derived from an intestinal endocrine tumor that developed in a double-transgenic mouse expressing the rat insulin promoter linked to the simian virus 40 large T antigen and the polyoma small T antigen (61). STC-1 cells (a gift from D. Hanahan, University of California at San Fransisco) were cultured in DMEM-F12 supplemented with 10% fetal bovine serum, L-glutamine, and antibiotics (100 U/ml penicillin plus 50 mmol/l streptomycin and gentamycin) in a humidified atmosphere with 10% CO₂ and 90% room air at 37°C. STC-1 cDNA was synthesized from 50–100 ng of poly A⁺-RNA (RNeasy, Qiagen; Valencia, CA) using a thermostable RT-PCR kit (Invitrogen; Carlsbad, CA).

RT-PCR using mRNA from STC-1 cells. Partial or full-length coding sequences of mT2Rs, PLC₂, TRPM4, TRPM5, Ca_v1.2, and Ca_v1.3 were amplified from cDNA using oligodeoxynucleotide primers designed for each specific mouse sequence, as listed in Table 1. PCR was performed in a total volume of 30 µl containing 100 ng of DNA, 300 nM of each primer in ExTaq buffer, and 2.5 units of ExTaq polymerase (TaKaRa; Madison, WI). An initial denaturation step of 94°C for 2 min was followed by 31 cycles of denaturation at 92°C for 40 s, annealing at 57°C for 40 s, and extension at 72°C for 2 min and finished with a final extension at 75°C for 5 min on a thermocycler (model PTC-200, MJ Research; San Francisco, CA). The housekeeping gene acetic ribosomal protein (ARP) was used as a control for cDNA quality and relative abundance. All PCR products were separated on 1% agarose gels and stained with ethidium bromide. Gel images were recorded from the UV illuminator and analyzed by imaging software (1D Image Analysis; Kodak, Rochester, NY). The predicted gene products were cloned into pCR II-TOPO vectors (Invitrogen), and their identities were confirmed by sequencing at least three positive clones.

Assay of [Ca²⁺]_i. [Ca²⁺]_i was measured by calcium fluorometry using fura-2 AM as previously described (56). Cells were grown on 9 x 22 mm glass coverslips in 35-mm dishes. The cells were washed twice with Hanks' balanced salt solution (HBSS; Gibco) supplemented with HEPES, pH 7.4, 1.26 mM CaCl₂, and 0.5 mM MgCl₂, 0.4 mM MgSO₄ and 0.1% BSA (referred as Ca²⁺ buffer) and were incubated at 37°C for 15 min in 1 ml of the same buffer with 1.0 µM fura-2 AM. The cultures were then washed three times with Ca²⁺ buffer and the coverslips inserted into a quartz cuvette containing 2 ml of Ca²⁺ buffer. The cuvette with the coverslip was placed into a Hitachi F-2000 fluorospectrophotometer. The incubation medium was continuously stirred at 37°C. The excitation wavelengths were set at 340 and 380 nm, and the emission wavelength was set at 510 nm. The maximum fluorescence was determined by injecting 100 µl of 5 mM digitonin into the cuvette, and the minimum fluorescence was measured after injection of 100 µl of 0.5 M EGTA, pH 8.0. A K_d of 224 nM was used for the Ca²⁺ dissociation constant from fura-2 in the cells at 37°C. [Ca²⁺]_i was determined automatically by the cation measurement software of the F-2000 fluorospectrophotometer.

For the Na⁺ replacement studies, STC-1 cells grown on coverslips were loaded with fura-2 and washed, as described above. Coverslips were washed once with Na⁺-free Ca²⁺ buffer (same as HBSS, except that NaCl, NaHCO₃, and Na₂HPO₄ were replaced by equal molarities of sucrose and ChCl and the pH was adjusted to 7.4 with KOH) and immediately inserted into the cuvette containing 2 ml of Na⁺-free buffer for the fluorometry studies.

Secretion of CCK from STC-1 cells. To determine the secretion of CCK from STC-1 cells, suspensions of these cells were plated at 1 x 10⁵ cells/cm² in 6-well plates (Costar 3516) and assays were performed on cultures that reached at least 80% confluency. Before treatment with agonists, tastants, and/or inhibitors, culture medium was removed and dishes were rinsed with HBSS adjusted to pH 7.4 and supplemented with 20 mM HEPES. Test agents dissolved in HBSS were then added immediately to the culture plate to give their final concentrations in a volume of 1 ml/well. Cells were then incubated at 37°C for various times (15–60 min). The medium was collected and centrifuged at 4°C for 5 min at 1,000 g to remove cell debris and the supernatants were stored at –20°C. CCK was measured by RIA using rabbit antiserum R016 raised against sulfated CCK-10 conjugated to keyhole limpet hemocyanin (a gift from Dr. T. E. Solomon). The detection limit and the ID₅₀ were 5 and 50 pM, respectively (72).

Materials. Tissue culture medium, fetal bovine serum and HBSS were purchased from Gibco (Grand Island, NY). Bitter tastant DB was from ICN Biomedicals (Aurora, OH) and PTC was from Sigma (St. Louis, MO). Channel blockers nitrendipine and glybenclamide were from Sigma, diltiazem was from Biomol Research Laboratories (Plymouth Meeting, PA). BAY K 8644, a voltage-sensitive Ca²⁺ channel activator, was from Biomol Research Laboratories. U-73122, an inhibitor of PLC₂ was from Calbiochem (BMD Biosciences, La Jolla, CA).

	RESULTS

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Expression of signal transducing proteins implicated in taste perception in enteroendocrine STC-1 cells. Our previous studies (74, 75) indicated that enteroendocrine STC-1 cells express transcripts encoding for several receptors of the T2R family and -subunits of gustducin and transducin (74, 75). Here, we extended these results showing that STC-1 cells express mT2R108, mT2R137, mT2R138, mT2R144, and mT2R135 (Fig. 1A). Furthermore, the results presented in Fig. 1A also show expression of G_gust, G3, G13, PLC2, in STC-1 cells, all of which have been associated with taste signaling (30, 81). We verified that 95% of the STC-1 cells used in the upcoming experiments were immunostained with antibodies directed against G_gust (Fig. 1B), indicating that most cells in the population express the -subunit of this G protein.

View larger version (65K): [in this window] [in a new window]   Fig. 1. A: expression of bitter taste receptors and signal transducing proteins in STC-1 cells. RT-PCR was performed using mouse-specific primers listed in Table 1 to detect the expression of functional murine type 2 family taste receptors (mT2Rs; i.e., mT2R108, mT2R137, mT2R138, mT2R144, and mT2R135) and the signal transducers -subunit of G protein gustducin (Ggust), G3, G13, and phospholipase C 2 (PLC2). Arrows indicate the corresponding PCR products, all of which were confirmed by sequencing. B: immunostaining of Ggust in the absence and presence of Ggust blocking peptide in STC-1 cells.

View larger version (22K): [in this window] [in a new window]   Fig. 2. The bitter stimuli denatonium benzoate (DB) and phenythiocarbamide (PTC) rapidly increase intracellular [Ca2+] ([Ca2+]i) in a dose-dependent manner in STC-1 cells. Cells grown on coverslips were washed and loaded with fura-2 AM. The cells on the coverslip were then washed and inserted into a quartz cuvette and placed into a Hitachi F-2000 fluorospectrophotometer, as described in MATERIALS AND METHODS. A–E: dose-dependent increases in [Ca2+]i concentrations induced by DB in STC-1 cells. Real-time measurement of [Ca2+]i after stimulation with increasing concentrations of DB (1 to 10 mM) was performed and the representative tracing of duplicate samples of 4 independent experiments was illustrated. F: peak increases in [Ca2+]i ([Ca2+]i) from baseline were measured in cells after treatment with DB at the indicated concentrations. Values are means ± SE of at least duplicate samples of four independent experiments (n = 4) and the statistical significance of the difference to controls (**P < 0.01) is indicated. G–I: dose-dependent increases in [Ca2+]i induced by PTC in STC-1 cells. Varying concentrations of PTC (2, 5, and 10 mM) were added at the time indicated by the arrow. After 75 s, the PTC-treated cells received a subsequent addition of DB (2 mM). [Ca2+]i was measured throughout the incubation period. PTC was dissolved in DMSO and the maximal final concentration of DMSO was 0.5% for the Ca2+ studies. J and K: STC-1 cells were pretreated with or without DB (2 mM) for 15 min during fura-2 loading. After being washed, cells were transferred to cuvette and were challenged with sequential addition of DB (2 and 8 mM) with 60 to 75 s apart. Traces show duplicate samples of 3 separate experiments.

To determine whether addition of DB to STC-1 cells attenuates the increase in [Ca²⁺]_i induced by a subsequent addition of DB (i.e., homologous desensitization), cells were exposed to 2 mM DB for 15 min and after being washed and loaded into the cuvette, cells were then challenged with two different doses of DB sequentially. As shown in Fig. 2, J and K, pretreatment of DB strikingly reduced the ability of a subsequent additions of DB at 2 and 8 mM to induce an increase in [Ca²⁺]_i in STC-1 cells. Our data suggest that like many other biological responses initiated by agonist binding to receptors and channels, DB exhibit the phenomenon of receptor-mediated adaptation or desensitization.

DB and PTC elicit rapid increase in [Ca²⁺]_i through Ca²⁺ influx. To determine the contribution of Ca²⁺ influx from the extracellular medium to the increase in [Ca²⁺]_i induced by DB or PTC in STC-1 cells, we prevented Ca²⁺ influx by chelating extracellular Ca²⁺ with EGTA and then sequentially challenged the cells with DB, PTC, and bombesin. As shown in Fig. 3, A and B, addition of EGTA blocked the increase in [Ca²⁺]_i induced by either DB or PTC but, in contrast, did not prevent the peak increase in [Ca²⁺]_i induced by bombesin, which is caused by mobilization of Ca²⁺ from intracellular stores (Fig. 3, C and D). EGTA (1.25 mM) completely blocked [Ca²⁺]_i increases elicited by 2 and 5 mM DB, but attenuated the peak increases of [Ca²⁺]_i stimulated by 10 mM DB to 25.2% ± 6.3% (means ± SE; n = 3) (Fig. 3, E and F). These results indicate that Ca²⁺ influx is responsible for a major component of the increase in [Ca²⁺]_i induced by DB or PTC in STC-1 cells.

View larger version (12K): [in this window] [in a new window]   Fig. 3. DB and PTC increase [Ca2+]i through Ca2+ influx in STC-1 cells. A–D: cells treated with either vehicle (VEH, water) or 1.25 mM EGTA for 50 s before stimulation with 5 mM PTC. After 80 s, either 2 mM DB (A and B) or 2.5 nM bombesin (BBS; C and D) was added. EGTA effect on higher concentrations of DB (5 and 10 mM in E and F) was examined. G and H: cells were preincubated with or without thapsigargin (50 nM) for 30 min during the fura-2 loading. Thapsigargin was added back to the cuvette after fura-2 was washed off, then DB (2 mM) or PTC (3 mM) was added at the indicated time points. Each tracing was a representative of duplicate samples of 3 separate experiments.

Inhibitors of L-type VSCCs prevent the increase in [Ca²⁺]_i induced by DB. Subsequent experiments were designed to identify the Ca²⁺ permeability pathways activated by bitter stimuli in STC-1 cells. L-type VSCCs mediate influx of extracellular Ca²⁺ into neuronal and neuroendocrine cells in response to membrane depolarization (5, 14). STC-1 cells have been shown to express functional L-type VSCCs that are opened by addition of KCl (42) but very little is known about the role of these channels in mediating responses initiated by bitter stimuli. Using RT-PCR, we verified that STC-1 cells express the pore-forming ₁ subunit isoforms Ca_v1.2 and Ca_v1.3 of L-type VSCCs, as shown in Fig. 4A. Accordingly, the addition of depolarizing concentrations of KCl (5–25 mM) to STC-1 cells induced a robust early spike in [Ca²⁺]_i, followed by a sustained plateau phase (Fig. 4, B–D). The increase in [Ca²⁺]_i induced by KCl was abrogated by chelation of extracellular Ca²⁺ by EGTA (Fig. 4, E and F) but it was unaffected by prior exposure to thapsigargin (Fig. 4, G and H).

View larger version (23K): [in this window] [in a new window]   Fig. 4. Bitter stimuli increase [Ca2+]i through Ca2+ influx mediated by the opening of L-type voltage-sensitive Ca2+ channels (VSCCs) in STC-1 cells. A: expression of the pore-forming 1 subunit isoforms Cav1.2 and Cav1.3 of L-type VSCCs in STC-1 cells. RT-PCR was carried out as described in MATERIALS AND METHODS using the primers described in Table 1. B–D: cells on the coverslips were exposed to increasing concentrations of KCl (5.0 to 25 mM) added to the cuvette, as indicated by the arrows. E and F: vehicle or EGTA (1.25 mM) was added to the cuvette for 50 s before the cells were sequentially stimulated with 25 mM KCl, 2 mM DB, and 2.5 nM BBS, as indicated. G and H: cells were treated with or without 50 nM thapsigargin for 30 min during fura-2 loading. After washing off the fura-2, cells were then inserted into the cuvette where the thapsigargin was also added back to the solution. KCl (25 mM) and 2.5 nM BBS were added, as indicated. Each trace was a representative of duplicate samples of 3 separate experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Inhibitors of L-type VSCCs prevent the increase in [Ca2+]i induced by DB or PTC. A–D: cells were pretreated with or without 1 µM nitrendipine for 30 min and 25 mM KCl, 3 mM PTC, 2 mM DB, and 2.5 nM BBS were added as indicated. Nitrendipine was dissolved in DMSO and final concentration for DMSO was 0.1%. E and F: STC-1 cells were pretreated without (E) or with (F) the L-type VSCC blocker diltiazem (0.1 mM) for 30 min. The same concentration of diltiazem was readded to the cells in F. DB (1 mM or 3 mM), and 2.5 nM bombesin (BBS) were added sequentially, as indicated. G and H: STC-1 cells were pretreated with or without either PDB (100 nM) for 10 min or PKC specific inhibitor Ro-31–8220 (3.5 µM) for 30 min during fura-2 loading. After fura-2 was washed off, cells were then inserted into the cuvette and treated with 3 mM DB as indicated. Each tracing was a representative of duplicate samples of 3 separate experiments.

To further substantiate that L-type VSCCs are involved in mediating the increase in [Ca²⁺]_i elicited by DB or PTC in STC-1 cells, we also determined whether the L-type VSCCs opener BAY K 8644 potentiates the increase in [Ca²⁺]_i induced by these bitter tastants. Addition of BAY K 8644 to STC-1 cells stimulated an increase in [Ca²⁺]_i in a concentration-dependent manner (Fig. 6, A–D). A detectable effect was seen at 0.3 µM and a robust elevation in [Ca²⁺]_i was obtained at either 3 or 10 µM. Interestingly, prior exposure to low concentrations of BAY K 8644 potentiated the ability of DB to elicit an increase in [Ca²⁺]_i. As shown in Fig. 6, E–G, addition of BAY K 8644 at 0.3–1 µM strikingly enhanced the increase in [Ca²⁺]_i induced by a subsequent addition of 1 mM DB, a concentration that induced a modest and slower increase in [Ca²⁺]_i, as shown previously in Fig. 2A. Collectively, the results presented in Figs. 4–6 demonstrate that the bitter compounds DB and PTC induce a striking increase in [Ca²⁺]_i through the opening of L-type VSCCs that mediate Ca²⁺ influx into STC-1 cells. In addition, the small increase in [Ca²⁺]_i originally elicited by either very low concentrations of BAY K 8644 or PTC primed subsequent challenge by low concentration of DB (1 mM) to a robust elevation through a mechanism of Ca²⁺ entry into STC-1 cells, possibly the opening of L-type VSCCs.

View larger version (14K): [in this window] [in a new window]   Fig. 6. The L-type VSCCs opener BAY K 8644 increases [Ca2+]i and potentiates the increase in [Ca2+]i induced by DB. A–D: addition of increasing concentrations of BAY K 8644 (0.3 µM to 10 µM) to fura-2 loaded STC-1 cells elicited a dose-dependent increase in [Ca2+]i. E–G: low concentrations of BAY K 8644 (0.1 to 1.0 µM) were added to the cuvette for 65 s and followed by 1 mM DB, as indicated. BAY K 8644 was dissolved in absolute ethanol and the final concentration for ethanol was 0.5%. Each trace represents duplicate samples of 4 separate experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Expression of TRPM4 and TRPM5 in STC-1 cells and requirement of Na+ for the increase in [Ca2+]i stimulated by DB or PTC in STC-1 cells. A, inset: RT-PCR of mRNA from STC-1 cells revealed the expression of TRPM4 and TRPM5. Arrows indicate the confirmed PCR products corresponding to the predicted sequences of TRPM4 and TRPM5. A–F: STC-1 cells were grown on coverslips, fura-2 loaded, and washed as described in Fig. 2. Coverslips were quickly washed once in Na+-free media and placed into the cuvette with 2 ml of a solution with (control; A, C, and E) or without Na+ (Na+ free; B, D, and F), as described in MATERIALS AND METHODS. The cultures were challenged with 2 mM DB, 3 mM PTC or 25 mM KCl, as indicated. Each trace represents duplicate samples of 3 separate experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 8. U-73122 prevented the increase in [Ca2+]i induced by DB or BBS, whereas glybenclamide did not affect basal or stimulated [Ca2+]i in response to DB, KCl, or BBS. A–C: cells were pretreated with or without 5 or 10 µM of the PLC inhibitor U-73122 for 30 min, and subsequently, 2 and 8 mM DB and 2.5 nM BBS were added, as indicated. U-73122 was dissolved in DMSO and final concentration for DMSO was 0.1%. D–F: glybenclamide (50 µM) was added to cells for 60 s, followed by addition of DB, KCl (20 mM), or BBS (2.5 nM). Glybenclamide was dissolved in DMSO and the final concentration for DMSO was 0.5%. Each tracing was a representative of duplicate samples of three separate experiments (n = 3).

CCK secretion from STC-1 cells in response to bitter tastants. STC-1 cells are known to produce and release CCK in response to multiple stimuli. We verified that 95% of the STC-1 cells used in our experiments were immunostained with antibodies directed against CCK (data not shown), indicating that virtually all the cells in the population produce this gastrointestinal hormone.

To determine the effect of DB on the secretion of CCK from STC-1 cells, cultures of these cells were washed with HBSS adjusted to pH 7.4 and incubated in this solution at 37°C for various times (15–60 min) in the absence or in the presence of DB at 5 mM. As shown in Fig. 9A, DB induced a marked increase in CCK release from STC-1 cells, compared with cells treated with the solvent control. In other experiments, we demonstrated that 5 mM DB stimulated CCK release to a degree comparable to that produced by 100 nM bombesin, a potent stimulus of CCK release in STC-1 cells (data not shown). In addition, DB increased CCK output into the medium in a dose-dependent manner in STC-1 cells (Fig. 9B).

View larger version (14K): [in this window] [in a new window]   Fig. 9. CCK release from STC-1 cells in response to DB is mediated by L-type VSCCs. CCK secretion was measured as described in MATERIALS AND METHODS. A: time course of DB-induced CCK-8 release from STC-1 cells. Before stimulation with DB, medium was removed and cultures were rinsed with Hanks' balanced salt solution (HBSS) adjusted to pH 7.4 and supplemented with 20 mM HEPES. DB dissolved in HBSS was then added to give a final concentration of 5 mM. Cells were then incubated at 37°C for various times (15–60 min). The medium was collected and centrifuged at 4°C for 5 min at 1,000 g to remove cell debris and the supernatants were stored at –20°C. The CCK concentrations in the media were determined by RIA. B: dose response of DB on CCK-8 release in STC-1 cells. For the dose response, increasing concentrations of DB were added to the cultures for 45 min and the media was then collected for CCK measurements. C: exposure to EGTA or to the L-type VSCCs blocker nitrendipine prevents CCK-8 release from STC-1 cells. Cells were pretreated in the absence (DMSO) or in the presence of 1 µM nitrendipine for 30 min or 1.25 mM EGTA for 2 min. After 5 mM DB was added, cultures were incubated at 37°C for 30 min. Culture media were then collected for the measurement of CCK by RIA.

	DISCUSSION

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Molecular sensing of the luminal contents of the GI tract not only regulates motility, release of GI hormones, and pancreatobiliary secretion, but it is also responsible for the detection of ingested drugs and toxins thereby initiating responses critical for survival. The enteroendocrine cells have been thought to play a critical role in the integration and coordination of these physiological responses but the initial molecular recognition events that sense the chemical composition of the luminal contents have remained elusive.

In view of the importance of chemical sensing in the regulation of food intake, digestion, and poison rejection, we started to determine whether bitter taste receptors are expressed in the GI tract. Our previous studies identified transcripts encoding members of the T2R family of bitter taste receptors in the gastric mucosa as well as in the lining of the intestine of mice and rats and in cultured enteroendocrine cells (74, 75). In the present study, we extended our previous findings demonstrating the expression of additional T2R receptors and effectors implicated in intracellular taste signal transduction, namely G_gust, PLC₂, and TRPM5 in STC-1 cells.

The complex pathways that mediate taste signaling in taste cells of the lingual epithelium are becoming increasingly understood (24, 44, 56) but it is not known whether these pathways operate in GI endocrine cells. In recent years, STC-1 cells have emerged as a valuable model system for studying the regulation of GI peptide synthesis and release from intestinal endocrine cells. STC-1 cells synthesize and store CCK and release this hormonal peptide in response to bombesin (10, 54, 67), pituitary adenylate cyclase-activating polypeptide (7), leptin (22), fatty acids (8, 66), orexin (36), aromatic amino acids (10), and peptidomimetic compounds (50, 53). STC-1 cells have been also used as a model for studies of enteroendocrine cell differentiation (59), GI peptide processing (79, 80) and regulation of CCK, glucose-dependent insulinotropic polypeptide, and proglucagon gene expression (2, 20, 31, 37, 58, 78). In this study, we demonstrate that the bitter tastants DB and PTC induced a marked increase in [Ca²⁺]_i in dose- and time-dependent fashion in STC-1 cells. In contrast, these tastants did not induce any detectable change in [Ca²⁺]_i in multiple cell lines that do not express T2Rs and G proteins implicated in bitter taste reception or GI peptides, including mouse Swiss 3T3 fibroblasts, rat intestinal epithelial IEC-18 and IEC-6 cells and human colonic (T84), pancreatic (BxPC3), or kidney (HEK-293) cells (74, 75). These findings are consistent with the notion that the effects on [Ca²⁺]_i signaling elicited by bitter stimuli in STC-1 cells are mediated by specific transducers that are expressed in these GI cells. We produced several lines of evidence indicating that either DB or PTC increases Ca²⁺ influx into SCT-1 cells. For example, chelating extracellular Ca²⁺ with EGTA blocked the increase in [Ca²⁺]_i induced by DB and PTC but in contrast, did not prevent the effect induced by the Ca²⁺-mobilizing neuropeptide bombesin. Reciprocally, thapsigargin, a compound that depletes the intracellular stores of Ca²⁺, blocked the transient increase in [Ca²⁺]_i induced by bombesin, but did not attenuate the [Ca²⁺]_i increase elicited by DB or PTC. These results indicate that Ca²⁺ influx mediates a substantial component of the increase in [Ca²⁺]_i induced by DB or PTC in STC-1 cells.

L-type VSCCs mediate influx of extracellular Ca²⁺ into neuronal and neuroendocrine cells in response to membrane depolarization (5, 14, 40). Accordingly, enteroendocrine STC-1 cells have been shown to express functional L-type VSCCs that are opened by addition of KCl (42). Using RT-PCR, we demonstrated that STC-1 cells express the pore-forming 1 subunit isoforms Ca_v1.2 and Ca_v1.3 of L-type VSCCs and verified that cell depolarization by KCl or addition of the L-type VSCC opener BAY K 8644 mimicked the increase in [Ca²⁺]_i induced by DB or PTC. Consequently, we determined whether the opening of L-type VSCCs could mediate the increase in [Ca²⁺]_i elicited by bitter stimuli in STC-1 cells. A salient feature of the results presented here is that treatment with the L-type VSCC blockers nitrendipine or diltiazem profoundly inhibited the increase in [Ca²⁺]_i elicited by tastants and KCl, indicating that the opening of these channels plays a major role in mediating the increase in [Ca²⁺]_i in response to DB or PTC in STC-1 cells. In line with this conclusion, exposure to BAY K 8644 potentiated the increase in [Ca²⁺]_i elicited by DB. Thus our results demonstrate for the first time that bitter tastants increase [Ca²⁺]_i through Ca²⁺ influx mediated by the opening of L-type VSCCs in enteroendocrine STC-1 cells.

Having demonstrated robust increases in [Ca²⁺]_i in STC-1 cells challenged with bitter tastants, we assessed whether these compounds also stimulate the release of CCK from these cells. Our results show that DB is a potent stimulant of CCK release from enteroendocrine STC-1 cells and that treatment with either EGTA or nitrendipine prevented this effect. We conclude that tastant-elicited CCK release is also mediated by an increase in [Ca²⁺]_i produced by the opening of L-type VSCCs.

We found that DB-induced Ca²⁺ influx required the presence of Na⁺ in the medium while KCl, which directly depolarizes the cells by reducing the outward gradient of K⁺, was still effective in stimulating Ca²⁺ influx even in the absence of extracellular Na⁺. These results imply that bitter stimuli stimulate Ca²⁺ influx into STC-1 cells through PLC- and Na⁺-dependent steps, possibly mediated by TRPM5 and TRPM4. Recent studies, using genetically modified mice, demonstrated that TRPM5 plays a critical role in the transduction of bitter stimuli in taste cells. Interestingly, this channel was expressed strongly not only in taste cells of the lingual epithelium but also in gastrointestinal cells. Taking together, these experiments suggest that DB and PTC induce a complex signaling cascade in enteroendocrine STC-1 cells leading to CCK release that appears to involve T2Rs, PLC, Na⁺, and Ca²⁺ influx and a PKC feedback loop, as presented schematically in Fig. 10. In agreement with this model, a previous study (69) indicated that DB does not exert any direct effect on L-type VSCC activity. It is interesting that the signaling cascade proposed in Fig. 10 can be distinguished from the bombesin-induced Ca²⁺ mobilization from thapsigargin-sensitive internal stores in the same STC-1 cells. There is increasing evidence indicating important differences between local and global changes in [Ca²⁺]_i response to different pools of Ins(1,4,5)-trisphosphate in a variety of cell types (17, 18, 76). It is therefore plausible that local changes in [Ca²⁺]_i in response to -mediated PLC2 stimulation by bitter stimuli activate TRPM4 and TRPM5 channels that mediate Na⁺-dependent depolarization leading to the activation of L-type VSCCs, as proposed in Fig. 10, while bombesin receptor activation, which is known to elicit Gq-mediated PLC₁ activation, leads to an increase in [Ca²⁺]_i that is not sufficient to activate TRPMs that mediate membrane depolarization and trigger L-type VSCC activation. Further experimental work is warranted to elucidate the differences between the signaling pathways triggered by taste receptors and hormonal neurotransmitters.

View larger version (32K): [in this window] [in a new window]   Fig. 10. Putative pathways triggered by bitter stimuli in GI enteroendocrine cells. The signaling cascade triggered by bitter stimuli in enteroendocrine STC-1 cells leading to CCK release, appears to involve T2Rs, PLC, Na+, and Ca2+ influx and a negative PKC feedback loop. This model proposes a mechanism by which bitter stimuli elicit responses in GI enteroendocrine cells. IP3, inositol trisphosphate; DAG, diacylglycerol; PKD, protein kinase D; PIP2, phosphatidylinositol (4,5) bisphosphate.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-55003 and DK-56930 (to E. Rozengurt).

	ACKNOWLEDGMENTS

 
E. Rozengurt is the Ronald S. Hirshberg Professor of Pancreatic Cancer Research.

	FOOTNOTES

 

Address for reprint requests and other correspondence: E. Rozengurt, 900 Veteran Ave., Warren Hall, Rm. 11-115, Dept. of Medicine, David Geffen School of Medicine, Univ. of California at Los Angeles, Los Angeles, CA 90095-1786 (e-mail: erozengurt{at}mednet.ucla.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.

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