Additive Effects of L-Ornithine on Preferences to Basic Taste Solutions in Mice

Abstract

In addition to the taste receptors corresponding to the six basic taste qualities-sweet, salty, sour, bitter, umami, and fatty-another type of taste receptor, calcium-sensing receptor (CaSR), is found in taste-bud cells. CaSR is called the 'kokumi' receptor because its agonists increase sweet, salty and umami tastes to induce 'koku', a Japanese word meaning the enhancement of flavor characters such as thickness, mouthfulness, and continuity. Koku is an important factor for enhancing food palatability. However, it is not well known whether other kokumi-receptors and substances exist. Here, we show that ornithine (L-ornithine but not D-ornithine) at low concentrations that do not elicit a taste of its own, enhances preferences to sweet, salty, umami, and fat taste solutions in mice. Increased preference to monosodium glutamate (MSG) was the most dominant effect. Antagonists of G-protein-coupled receptor family C group 6 subtype A (GPRC6A) abolished the additive effect of ornithine on MSG solutions. The additive effects of ornithine on taste stimuli are thought to occur in the oral cavity, and are not considered post-oral events because ornithine's effects were confirmed in a brief-exposure test. Moreover, the additive effects of ornithine and the action of the antagonist were verified in electrophysiological taste nerve responses. Immunohistochemical analysis implied that GPRC6A was expressed in subsets of type II and type III taste cells of mouse circumvallate papillae. These results are in good agreement with those reported for taste modulation involving CaSR and its agonists. The present study suggests that ornithine is a kokumi substance and GPRC6A is a newly identified kokumi receptor.

Keywords: GPRC6A; L-ornithine; basic taste solutions; chorda tympani; electrophysiology; immunohistochemistry; kokumi; taste preference.

Figure 1

Two-bottle preference test using miso soup or a taste mixture in the presence or absence of corbiculae, L-ornithine (L-Orn), or D-ornithine (D-Orn). Fluid intake (mean ± SE; n = 8) is compared between miso soup and miso soup with corbiculae (A), L-Orn (B), or D-Orn (C), and between a taste mixture with or without L-Orn (D). The taste mixture is an aqueous solution consisting of 0.05 M MSG, 0.05 M sucrose, and 0.05 M NaCl. *** p < 0.001.

Figure 2

Additive effects of different concentrations of L-ornithine (Orn). (A) Water intake with or without five concentrations of Orn. (B) Preference score for water with Orn. (C) Intake of a mixture of 0.05 M MSG, 0.05 M sucrose, and 0.05 M NaCl with or without Orn. (D) Preference scores for the mixture with Orn. Each value is mean ± SE; n = 8. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Additive effects of 1 mM L-ornithine (Orn) on different concentrations of taste solutions. Fluid intake (mean ± SE; n = 8) with and without Orn is shown for MSG (A), MPG (B), MSG with 0.01 mM amiloride (C), sucrose (D), NaCl (E), Intralipos (F), quinine hydrochloride (QHCl) (G), citric acid (H), and IMP (I). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Additive effects of different concentrations of L-ornithine (Orn) on a fixed concentration of umami solutions. Preference scores (mean ± SE; n = 8) are shown for 0.01 M IMP with Orn (A), 0.05 M MSG with Orn (B), 0.05 M MPG with Orn (C), and a mixture of 0.05 M MPG and 0.05 M NaCl with Orn (D). * p < 0.05, ** p < 0.01 (vs. preference score when supplemented with 0.3 mM Orn).

Figure 5

Effects of GPRC6A antagonists on the amount of intake of 0.05 M MSG with and without 1 mM L-ornithine (Orn). Each of the antagonists, (A) NPS-2143, (B) Calindol and (C) EGCG, dose-dependently suppressed. Each of the antagonists (NPS-2143, calindol and EGCG) dose-dependently suppressed Orn-induced preference. Each value is mean ± SE; n = 8. *** p < 0.001.

Figure 6

Brief-exposure (5 min) two-bottle preference test for 0.05 M MSG with and without 1 mM L-ornithine (Orn) and effects of EGCG. Intake of MSG increased by addition of Orn in the absence of the GPRC6A antagonist (A), or in the presence of 10 µM EGCG (B), 100 µM EGCG (C); however, the effect of Orn was blocked in the presence of 100 µM EGCG. Each value is mean ± SE; n = 8. * p < 0.05, ** p < 0.01.

Figure 7

Sample recordings of chorda tympani responses to taste stimuli with and without 1 mM L-ornithine (Orn). (A) Responses to 0.1 M NH₄Cl, (B) Responses to 0.05 M MSG, MSG + Orn, and MSG + Orn + 0.6 mM calindol. (C) Responses to 0.05 M sucrose with and without Orn. (D) 0.05M NaCl with and without Orn. (E) Responses to 10% Intralipos with and without Orn. (F) Responses to 0.02M QHCl with and without Orn. (G) Responses to 0.02M citric acid with and without Orn. (H) Responses to 0.01 M IMP with and without Orn. The horizontal bar indicates 30 s.

Figure 8

Quantitative representation of the magnitude of chorda tympani responses. (A) A concentration-response curve of the effects of aqueous L-ornithine (Orn) solutions on nerve responses. (B) The sum of the individual responses to 0.05 M MSG and 1 mM Orn, the response to a binary mixture of 0.05 M MSG and 1 mM Orn, and a mixture of MSG, Orn and 0.6 mM calindol. (C) The sum and mixture of 0.05 M sucrose and Orn. (D) The sum and mixture of 0.05M NaCl and Orn. (E) The sum and mixture of 10% Intralipos and Orn. (F) The sum and mixture of 0.02 M QHCl and Orn. (G) The sum and mixture of 0.02 M citric acid and Orn. (H) The sum and mixture of 0.01 M IMP and Orn. Each value is mean ± SE; n = 5. * p < 0.017 (after Bonferroni correction) for (B), * p < 0.05, ** p < 0.01 (paired t-test, two-tailed) for (C–E). N.S., not significant.

Figure 9

Immunohistochemical analysis of GPRC6A in taste cells of mouse circumvallate papilla taste buds. (A,B) Fluorescence micrographs of the taste buds immunostained with the anti-GPRC6A antibody in the absence (A) or presence (B) of the antigen peptide (left panels; middle panels, Nomarski images of the left panels; right panels, merged images of respective right and middle panels). (C) Immunoreactivity for GPRC6A is expressed in a small subset of type II taste cells. Shown are immunoreactivity for GPRC6A (red, left panel), immunoreactivity for a-gustducin (green, middle panel) and a merged image of both panels (right panel). Arrowheads indicate GPRC6A-positive type II taste cells. (D) Immunoreactivity for GPRC6A is expressed in a subset of type III taste cells. Shown are immunoreactivity for GPRC6A (red, left panel), immunoreactivity for SNAP-25 (green, middle panel) and a merged image of both panels (right). Arrowheads indicate GPRC6A-positive type III taste cells. Scale bars, 20 mm.