Expression of bitter taste receptors of the T2R family in the gastrointestinal tract and enteroendocrine STC-1 cells

Abstract

Although a role for the gastric and intestinal mucosa in molecular sensing has been known for decades, the initial molecular recognition events that sense the chemical composition of the luminal contents has remained elusive. Here we identified putative taste receptor gene transcripts in the gastrointestinal tract. Our results, using reverse transcriptase-PCR, demonstrate the presence of transcripts corresponding to multiple members of the T2R family of bitter taste receptors in the antral and fundic gastric mucosa as well as in the lining of the duodenum. In addition, cDNA clones of T2R receptors were detected in a rat gastric endocrine cell cDNA library, suggesting that these receptors are expressed, at least partly, in enteroendocrine cells. Accordingly, expression of multiple T2R receptors also was found in STC-1 cells, an enteroendocrine cell line. The expression of alpha subunits of G proteins implicated in intracellular taste signal transduction, namely Galpha(gust), and Galpha(t)-(2), also was demonstrated in the gastrointestinal mucosa as well as in STC-1 cells, as revealed by reverse transcriptase-PCR and DNA sequencing, immunohistochemistry, and Western blotting. Furthermore, addition of compounds widely used in bitter taste signaling (e.g., denatonium, phenylthiocarbamide, 6-n-propil-2-thiouracil, and cycloheximide) to STC-1 cells promoted a rapid increase in intracellular Ca(2+) concentration. These results demonstrate the expression of bitter taste receptors of the T2R family in the mouse and rat gastrointestinal tract.

Figure 1

Expression of Gα_gust and G_t-2 in rat GI tissues and a gastric endocrine cell cDNA library. Consensus primers to amplify Gα_gust and Gα_t-2 were designed based on the published rat, mouse, and human sequences, as shown in Table 1. PCR amplification was performed on reverse-transcribed poly(A)⁺ RNA isolated from rat antrum (A), fundus (F), and duodenum (D), and on cDNA from a gastric endocrine cell library (GEC). The predicted sizes of the PCR products of Gα_t-2 (Top) and Gα_gust (Middle) are 340 bp and 332 bp, respectively. A cDNA fragment (764 bp) corresponding to β-actin was amplified as a control transcript from the respective cDNA sample (Bottom).

Figure 2

Immunostaining of Gα_t-2 and Gα_gust in mouse fundus (A and B) and antrum (C and D). (A) Immunostaining with antibody against Gα_t-2 The base of the fundic glands is rich in positively stained cells (arrows) (×40). (Inset) Higher-power (×100) view of a gland showing that the stained cell is round, with a central or slightly eccentric nucleus and pale cytoplasm. (B) A consecutive section stained with antibody against Gα_gust. There are no positive cells at this portion of the gland (×40). (C) Antral mucosa immunostained with antibody against Gα_t-2. There are no positive cells (×40). (D) A consecutive section stained with antibody against Gα_gust. Arrows point at some of the many positively stained cells (×40). (Inset) Higher-power (×100) view of a Gα_gust-positive cell exhibiting an elongated shape with a luminal pole and a projection toward the basement membrane.

Figure 3

Expression of members of the rT2R family of receptors in rat gastric and duodenal mucosa. RT-PCR was performed by using specific primers for each of the 11 rT2R subtypes (Table 1) on poly(A)⁺ RNA isolated from rat antral, fundic, duodenal mucosa, and IEC-6 cells, and on cDNAs from a rat gastric endocrine cell cDNA library. PCR products were separated on 1% agarose gel containing ethidium bromide, and products of the predicted size (Table 1) for each rT2R subtype were subcloned and sequenced to verify their identity. Control transcript β-actin from the respective sample is shown in the far right lanes.

Figure 4

Distribution of mT2R19 transcripts in mouse tissues. RT-PCR using mT2R19-specific primers (Table 1) was performed on cDNAs prepared from various mouse tissues. PCR products were separated on 1% agarose gel containing ethidium bromide, and the identity of the predicted mT2R19 cDNA fragment (698 bp) was confirmed by DNA sequencing. A: antrum; F: fundus; D: duodenum; I: ileum; J: jejunum; C: colon; L: liver; H: heart; K: kidney; T: tongue (Upper). As a control, β-actin from the respective samples was amplified (Lower).

Figure 5

Expression of Gα_t-2, Gα_gust, and members of the T2R family in STC-1 cells. (A) RT-PCR analysis for the α subunits of G_t-₂ and G_gust was performed on poly(A)⁺ RNA isolated from STC-1 cells. PCR products with the predicted size were subcloned and sequenced to confirm their identity. (B) Immunoblot analysis for Gα_t-2 and Gα_gust was performed on total protein extracts prepared from STC-1 cells. Normal or preabsorbed Gα_t-₂ and Gα_gust-specific antibodies were used to detect the presence of these Gα subunits in STC-1 cell lysates by Western blotting. (C) RT-PCR analysis using rat T2R subtype-specific primers was performed on the same cDNAs used in the experiments described in A. PCR products corresponding to the predicted mT2Rs were subcloned and sequenced to confirm their relatedness to published rat and mouse sequences.

Figure 6

STC-1 cells respond to bitter tastant molecules with an increase in [Ca²⁺]_i. The [Ca²⁺]_i of individual cells was measured before and after exposure to single concentrations of bitter tastants. (A) Percent of cells that responded to each tastant: DB, denatonium benzoate at 10 mM (n = 160 cells), 1 mM (n = 98 cells), 0.1 mM (n = 82 cells); PTC, phenylathiocarbamide at 3 mM (n = 60 cells); 6-PTU, 6-n-propyl thiouracil at 1 mM (n = 63 cells); CAF, caffeine at 10 mM (n = 52); NIC, nicotine at 10 mM (n = 53 cells); CHL, chloroquine at 1 mM (n = 52 cells); CYC; cycloheximide at 50 μM (n = 538 cells). The values in parenthesis are the number of cells analyzed. (B) Typical individual trace of [Ca²⁺]_i from a single STC-1 cell exposed to 10 mM denatonium benzoate (DB). (C) Typical individual trace of [Ca²⁺]_i from a single IEC-6 cell exposed sequentially to 10 mM denatonium benzoate (DB) followed by 50 nM angiotensin II (Ang), added as a positive control. (D) Individual trace of [Ca²⁺]_i from a single STC-1 cell exposed to 50 μM cycloheximide (CYC) that displayed an oscillatory response. (E) Individual trace of [Ca²⁺]_i from a single STC-1 cell exposed to 250 μM cycloheximide (CYC) that displayed a peak and plateau response. The addition of the tastant or agonist is marked by the arrow.