Additive effects of different concentrations of ornithine (Orn).

(A) Water intake with or without five concentrations of Orn. (B) Intake of 0.03 M MSG with or without Orn. Each value represents the mean ± SEM; n = 6. **P < 0.01, ***P < 0.001.

Additive effects of 1 mM ornithine (Orn) on fluid intake for four different taste solutions.

Fluid intake (mean ± SEM; n = 8) with and without Orn is shown for IMP (A), MSG (B), MPG (C), Intralipos (D), sucrose (E), NaCl (F), citric acid (G), and quinine hydrochloride (QHCl) (H). *P < 0.05, **P < 0.01, ***P < 0.001.

Brief exposure (10 min) two-bottle preference test for different liquids with and without 1 mM ornithine (Orn) and effects of calindol and EGCG, antagonists of GPRC6A.

(A) Intake of water with and without Orn. (B) Intake of 0.03 M MSG with and without Orn. (C) Intake of MSG with and without Orn in 60 µM calindol. (D) Intake of MSG with and without Orn in 300 µM calindol. (E) Intake of MSG with and without Orn in 30 µM EGCG. (F) Intake of MSG with and without Orn in 100 µM EGCG. Each value represents the mean ± SEM; n = 7. *P < 0.05, **P < 0.01.

Effects of bilateral chorda tympani transection (xCT) and control sham operation (Sham OP) on the intake of 0.03 M MSG with and without 1 mM ornithine (Orn).

Fluid intake was compared before (A, C) and after (B, D) the operations. Each value represents the mean ± SEM; n = 4. **P < 0.01.

Sample recordings of chorda tympani (CT) responses and quantitative representation of the mean magnitude of CT responses.

(A) Nerve responses to five concentrations of ornithine (Orn). (B) Nerve responses to 0.03 M MSG with and without 1 mM Orn in water and in 0.01 mM amiloride, a sodium channel blocker. (C) Nerve responses to MSG, MSG with Orn, and MSG with Orn in 300 µM calindol. (D, E, F) Graphical representations of the mean magnitude of CT responses corresponding to A, B, C, respectively. Each value represents the mean ± SEM, normalized to the response to 0.1 M NH4Cl = 1.0; n = 4 or 5. *P < 0.05, **P < 0.01, ***P < 0.001.

Immunohistochemical localization of GPRC6A in rat taste papillae.

(A) A significant number of spindle-shaped taste cells exhibited intense GPRC6A-immunoreactivity in the fungiform papilla. (B, C) GPRC6A-immunopositive taste cells were barely detectable in the foliate (B) and circumvallate (C) papillae. Left panels show GPRC6A in red, middle panels show Nomarski images of the left panels, and right panels show merged images of respective right and middle panels. Scale bars: 50 µm.

Immunohistochemical analysis of colocalization of GPRC6A-immunoreactivity and cell type-specific markers in taste cells of rat fungiform papilla taste buds.

(A) Some, but not all, GPRC6A-immunopositive cells exhibited immunoreactivity for IP3R3, a marker for the majority of the type II cell population. Arrow indicates a GPRC6A/IP3R3 double-positive taste cell. (B) α-Gustducin, another marker for a subset of type II cells, was unlikely to be colocalized with GPRC6A in single taste cells. (C, D) Neither 5-HT (C) nor SNAP25 (D), both are specific markers for type III taste cells, were likely to be colocalized with GPRC6A in single taste cells. Scale bars, 10 µm.

Effects of ornithine (L-ornithine, Orn) supplementation in low sodium (0.7%) miso soup on three kokumi attributes: thickness, mouthfulness, and continuity in humans.

The addition of ornithine at three concentrations (1, 3, and 10 mM, labeled as T-1, T-2, and T-3, respectively) increased these three attributes in a dose-dependent manner. Palatability also improved in a dose-dependent manner. All values for kokumi attributes and palatability were set to 0 for the control miso soup without ornithine (C). Each value represents the mean ± SEM (n = 17).

Long-term exposure (one day) two-bottle preference test for different liquids with and without 1mM gallate (ethyl gallate, Kanto Chemical, Toko, Japan) and effects of 100 µM epigallocatechin gallate (EGCG), an antagonist of GPRC6A.

(A) Intake of water (W) with and without gallate (G), (B) Intake of 0.03M MSG (M) with and without gallate. (C) Comparison between MSG + EGCG and MSG + gallate + EGCG, (D) Intake of 0.01mM quinine-HCl (Q) with and without gallate, (E) Comparison between Q + EGCG and Q + gallate + EGCG. Each value represents the mean ± SEM (n = 8). *P<0.05, **P<0.01 (paired t-test, two-tailed). These results show that gallate itself is not palatable, however, it increases the preference for MSG and QHCl and an antagonist of GPRC6A eliminates these preferences.

Immunohistochemical localization of GPRC6A and CaSR (calcium-sensing receptor) in rat fungiform taste papillae.

GPRC6A and CaSR are basically expressed in distinct subpopulations of fungiform taste cells. Nomarski image of the tissue section examined is shown in the rightmost panel. Scale bar, 10 µm. The procedures for immunohistochemistry were essentially the same as those described in the Materials and methods section. For CaSR staining, the mouse anti-CaSR antibody (1:100; ab19347, Abcam) was used.