Caffeine induces gastric acid secretion via bitter taste signaling in gastric parietal cells

Abstract

Caffeine, generally known as a stimulant of gastric acid secretion (GAS), is a bitter-tasting compound that activates several taste type 2 bitter receptors (TAS2Rs). TAS2Rs are expressed in the mouth and in several extraoral sites, e.g., in the gastrointestinal tract, in which their functional role still needs to be clarified. We hypothesized that caffeine evokes effects on GAS by activation of oral and gastric TAS2Rs and demonstrate that caffeine, when administered encapsulated, stimulates GAS, whereas oral administration of a caffeine solution delays GAS in healthy human subjects. Correlation analysis of data obtained from ingestion of the caffeine solution revealed an association between the magnitude of the GAS response and the perceived bitterness, suggesting a functional role of oral TAS2Rs in GAS. Expression of TAS2Rs, including cognate TAS2Rs for caffeine, was shown in human gastric epithelial cells of the corpus/fundus and in HGT-1 cells, a model for the study of GAS. In HGT-1 cells, various bitter compounds as well as caffeine stimulated proton secretion, whereby the caffeine-evoked effect was (i) shown to depend on one of its cognate receptor, TAS2R43, and adenylyl cyclase; and (ii) reduced by homoeriodictyol (HED), a known inhibitor of caffeine's bitter taste. This inhibitory effect of HED on caffeine-induced GAS was verified in healthy human subjects. These findings (i) demonstrate that bitter taste receptors in the stomach and the oral cavity are involved in the regulation of GAS and (ii) suggest that bitter tastants and bitter-masking compounds could be potentially useful therapeutics to regulate gastric pH.

Keywords: TAS2Rs; bitter taste receptors; caffeine; gastric acid secretion; homoeriodictyol.

Fig. 1.

Results of the gastric pH measurements demonstrate that the effect of caffeine (CAF) on reacidification time is influenced by the type of administration. (A) Overview of the different administration types in the human intervention trial. (B) Overview of the study procedure. (C) Gastrograms of different Heidelberg capsule measurements from one test subject combined in one graphic show that 150 mg caffeine diluted or administered with 125 mL water (blue line) administered by sip and spit (3) prolongs the reacidification time (i.e., time until the original pH is reached again) more than administration via drinking (2) or in encapsulated form (1). (D) Delta reacidification time of gastrograms show that sip-and-spit administration resulted in the highest prolongation of reacidification time compared with gastric and gastric plus oral administration. (E) Delta slope of the gastrograms indicate that encapsulated administration (gastric delivery) strongly stimulate GAS when reacidification has started. Data are displayed as mean ± SEM, n = 5–10; one-way ANOVA with Holm–Šídák post hoc test; significant (P < 0.05) differences are indicated by distinct letters [*P < 0.05, significant vs. water control (basal = 0) tested with paired Student t test].

Fig. 2.

Addition of HED reduces the caffeine-evoked effects on reacidification time or the slope in gastric pH measurements via administration by drinking 150 mg caffeine (CAF) with or without 30 mg HED dissolved in 125 mL water (A–C) or by encapsulated test compounds in combination with 125 mL water 25 min before alkaline challenge (D–F). (A and D) Gastrograms of Heidelberg capsule measurements according to the three different delivery protocols from one test subject are presented in one graphic. (B and E) Delta reacidification time of gastric pH measurements in subjects after consumption of CAF or CAF plus HED (basal = 0). (C and F) Delta slope of gastric pH measurements in subjects after consumption of CAF or CAF plus HED (basal = 0). Data are displayed as mean ± SEM: (B and C) CAF, n = 10; CAF plus HED, n = 6; (E and F) CAF, n = 7; CAF plus HED, n = 6 (*P < 0.05 and **P < 0.01 indicate significant differences by Student’s t test).

Fig. S1.

(A) Impact of 1 mM NaHED on gastric motility in the human stomach in the fundus region. Data represent the percentage change of tension before and after adding NaHED. Cholinergically mediated contractions of the tissue were evoked by EFS (200 mA for 0.5 ms at 5 Hz for 10 s every 1 min). Maximum relaxation was detected after 40 min incubation time. (B) Representative traces of the vehicle control and the trace incubated with 1 mM NaHED. Data are given as mean ± SEM. Statistics: vehicle control, n = 2; NaHED, n = 3; t test vs. vehicle control, *P < 0.05.

Fig. S2.

(A) Bitter intensities of 1,200 mg/L caffeine and 1,200 mg/L caffeine in combination with 240 mg/L HED were assessed in 13 sensorial untrained test subjects under colored light, repeated three or four times. Statistics derived by Student’s t test, **P < 0.01. (B) Reacidification time, measured by the Heidelberg detection system, of different concentrations of caffeine and 125 mL water administered by drinking, allowing activation of oral and gastric TAS2Rs, in comparison with 125 mL water alone. Statistics: Student’s t test, 150 mg caffeine vs. water. (C) Spearman correlation analysis between caffeine bitter intensity and reacidification time after administration of 150 mg caffeine via the drinking protocol.

Fig. 3.

(A, a–p) Immunochemical localization of TAS2R10 and GNAT2 in (A) gastric tissue and (B) HGT-1 cells with and without preincubation with a blocking peptide. (a) In the gastric corpus/fundus, cytoplasmic reactivity of TAS2R10 in parietal and chief cells (one arrow) was detected whereas foveolar cells were negative (two arrows). Detail (b) shows parietal and chief cells. In the gastric antrum (e and f), very faint cytoplasmic and focal membranous reactivity of TAS2R10 in glandular cells was detected (one arrow). Foveolar cells are negative (two arrows). (f) Detail showing glandular cells. GNAT2 was localized in the gastric fundus (i and j) parietal and chief cells (one arrow, j). Foveolar cells demonstrate membranous staining (two arrows, j). (m and n) In gastric antrum, membranous reactivity of GNAT2 in glandular cells (one arrow, m and n) was detected whereas foveolar cells were negative (two arrows, m). (c, d, g, h, k, l, o, and p) Corresponding negative controls. (B) Staining of HGT-1 cells with TAS2R10 and GNAT2 antisera (green) with and without specific blocking peptide and cell-surface labeling with con A (red).

Fig. S3.

Immunocytochemical costaining patterns of anti-TAS2R10 and epitope tag-specific antibodies in HEK-293T-Gα16gust44 cells. Specific staining of HEK-293T-Gα16gust44 cells expressing TAS2R10 is demonstrated by the TAS2R10 antibody (green). TAS2R10 antibody blocked with specific blocking peptide showed no staining of cells expressing TAS2R10 or in cells expressing irrelevant target TAS2R16. The epitope-tagged receptor proteins were detected using an HSV-specific antiserum (red). Cell surface labeling (blue) was achieved by using con A.

Fig. S4.

Identification of human gastric cell types by H&E staining in gastric fundus showing localization of gastric cell types (A). Parietal cells are localized in the glands of gastric fundus and body and are scattered in the middle and, to a lesser extent, the bottom part of the mucosa. They are characterized by broad pink cytoplasms. Chief cells stain with basophilic cytoplasm and are mainly located in the bottom parts of the mucosa, which can be seen in more detail in the Inset: gastric glands with parietal (single arrow) and chief cells (double arrow). (B) Immunohistochemical localization of taste receptor TASR10.

Fig. S5.

Identification of human gastric cell types by H&E staining in gastric antrum (A). (Inset) Detail with gastric glands of antrum. (B) Immunohistochemical localization of taste receptor TASR10 in gastric glands at the bottom part of the mucosa.

Fig. 4.

Bitter tastants increase proton secretion in human gastric cells. Studies were performed with cultured HGT-1 cells loaded with the pH-sensitive fluorescent dye SNARF-1-AM and treated with test compounds for 10 min (A, B, and D). Results are presented as the IPX. A lower IPX value indicates increased proton secretion. Data displayed as mean IPX ± SEM. (A) IPX of HGT-1 cells after treatment with histamine (HIS; 1 mM), yohimbine (YO; 30 µM), denatonium benzoate (DB; 30 µM), caffeine (CAF; 3.0 mM), theobromine (TH; 0.3 mM), tannic acid (TA; 3 µM), aristolochic acid (AA; 0.3 µM), and sodium benzoate (SB; 3.0 mM) in comparison with untreated cells (i.e., control; marked as “C”) or 0.1% DMSO-treated cells [solvent control for yohimbine; n = 3–16; six technical replicates (tr)]. (B) Coadministration of HED reduces the stimulating effect of caffeine on proton secretion (n = 4–37; tr = 6). (C) Inhibition curves of TAS2R43 assessed through calcium imaging experiments in transfected HEK-293T cells. Cells were costimulated with 0.03 µM aristolochic acid (Arist. Ac.) or caffeine 1 mM and increasing concentrations of the inhibitors HED or ED. Caffeine and aristolochic response amplitudes (ΔF/F₀) were 0.14 and 0.39, respectively. Concentrations were chosen based on preliminary experiments to elicit the strongest effect. (D) IPX of HGT-1 cells transfected with nontargeting gRNA (NC) or HGT-1 cells with KO of TAS2R43 by CRISPR-Cas9 deletion treated with histamine (HIS; 1 mM), aristolochic acid (AA; 0.3 µM), caffeine (CAF; 3.0 mM), or 3.0 mM caffeine and 0.3 mM HED (n = 5–6; tr = 6). (E) Percentage inhibition of 3 mM caffeine effect on IPX of HGT-1 cells in comparison after treatment with 3 mM caffeine in combination with 0.3 mM HED, 5 µM U73122, 100 µM neomycin, or 30 µM NKY80 (n = 3–6; tr = 6). (F) cAMP concentration in HGT-1 cells after 10 min treatment with 3 mM caffeine, 0.3 mM HED, or in combination in comparison with DMEM, EtOH 0.1%, or forskolin 10 µM (n = 4; tr = 2). (A–E) Data presented as mean ± SEM. (C) Data presented as mean ± SD. Statistics: (A, B, and F) one-way ANOVA with Holm–Šídák post hoc test (F) vs. DMEM and (A and C–F) Student’s t test. Significant (P < 0.05) differences are indicated by letters or as follows: ^###P < 0.001 vs. DMSO 0.1%; ***P < 0.001, **P < 0.01, and *P < 0.05.

Fig. S6.

IPX of HGT-1 cells treated for 10 min with (A) caffeine in different concentrations (n = 5; tr = 6) (B) caffeine alone and in combination with the diluent for ED 1% EtOH. Histamine (HIS) 1 mM was used as positive control (n = 4–37; tr = 6). (C) Caffeine alone and in combination with two concentrations of ED; data displayed as mean ± SEM; n= 4–37; tr = 6, Statistics: (A) one-way ANOVA with Holm–Šídák post hoc test. Significant differences are indicated by ***P < 0.001 or **P < 0.01; *P < 0.05 vs. control. (B and C) One-way ANOVA on ranks with Dunn’s post hoc test. Significant differences are indicated by letters. The lower the IPX, the stronger the proton secretion.

Fig. S7.

(A) Location of the 13-bp deletion in the TAS2R43 gene in the HGT-1 TAS2R43-KO cells established with CRISPR-Cas9–directed KO in comparison with negative control (NC), cells transfected with a nontargeting gRNA, and WT (HGT-1-WT). These results derive from whole-genome sequencing aligned to HG19 and analyzed using IGV software. (B) Verification of deletion also on mRNA level by Sanger sequencing.