Figure 1.
(A) RT-PCR for CaSR expression in taste bud-enriched circumvallate (cv), foliate (foli) and non-taste bud (nt) lingual epithelium. nc - negative control (lacking template); M - molecular standard. (B–E) Immunostaining for CaSR in taste buds. CaSR immunofluorescence is seen in most circumvallate (B), foliate (C), palate (D) and fungiform (E) taste buds. Immunofluorescent images (green) were superimposed on DIC images. (F) Validating the anti-CaSR antibody. The CaSR antiserum was preabsorbed with an excess of antigen peptides. The circumvallate sections reacted with preabsorbed and non-absorbed antibodies and were processed simultaneously. Images were taken under the same illumination conditions and detector settings. Scale bars 20 µm.
Figure 2.
Confocal images showing colocalization of CaSR and the taste cell markers in taste cells from mouse circumvallate papillae.
(A–C) A longitudinal section of a circumvallate taste bud immunostained with antibodies against CaSR (A) and PLCβ2 (B). (C) Overlay of A and B. (D) A transverse section of a circumvallate taste bud immunostained with antibodies against CaSR (green) and PLCβ2 (red). (E–G) A longitudinal section of a circumvallate taste bud immunostained with antibodies against CaSR (E) and NCAM (F). (G) Overlay of E and F. (H) A transverse section of a circumvallate taste bud immunostained with antibodies against CaSR (green) and NCAM (red). Scale bars 20 µm. Arrowheads indicate double-labeled cells.
Figure 3.
Taste cell responses (ΔCa2+) evoked by kokumi stimuli, recorded in a slice preparation of the mouse circumvallate papilla.
(A) Taste cells were stimulated sequentially with three kokumi substances, cinacalcet (CCT, 10 µM), glutathione (GSH, 100 µM) and γ-glutamyl-valinyl-glycine (γEVG, 100 µM), as well as γEVG + NPS2143 (3 µM), a CaSR antagonist. Arrowheads below traces indicate the stimulation. (B) Concentration-response relationship for γEVG (mean ± SE; n = 4 cells). (C) Taste responses elicited by γEVG (100 µM) were inhibited by the CaSR antagonist, NPS2143 (3 µM), but the umami (MPG 100 mM + IMP 1 mM) and the sweet (SC45647, 10 µM) responses were unaffected. Mean amplitudes of γEVG-, MPG + IMP-, and SC45647-induced responses in the presence or absence of 3 µM NPS2143 are shown (mean ± SE; *p≤0.05, n = 4 cells). Raw traces are shown in A.
Figure 4.
Ca2+ response elicited by γEVG involves intracellular Ca2+ stores and phospholipase C.
(A) γEVG (100 µM) was focally applied in medium containing Ca2+ (left trace) or in the absence of Ca2+ (Ca-free medium with 0.2 mM EGTA; right trace). (B) Responses elicited by depolarization (bath-applied KCl, 50 mM) and influx of Ca2+ through voltage-dependent Ca2+ channels were abolished in the absence of extracellular Ca2+. (C) Mean amplitudes of responses in the presence or absence of Ca2+ in the medium (mean ± SE; *p≤0.05, n = 4 cells). (D) Responses to γEVG inhibited by U73122 (10 µM). (E) Mean amplitudes of the responses in the presence or absence of U73122 (mean ± SE; *p≤0.05, n = 4 cells).
Figure 5.
γEVG-responding taste cells are different from MPG + IMP-responding cells.
(A) Taste cell responses were recorded in lingual slice preparations of mouse circumvallate papilla. Preparations were sequentially stimulated with γEVG (100 µM) and MPG (100 mM) + IMP (1 mM). The traces show superimposed responses from γEVG-responding cells (A) and MPG + IMP-responding cells (B). Responses to γEVG were only observed in cells lacking response to MPG + IMP. (C) We recorded from 132 Calcium Green-loaded taste cells in 16 lingual slices. Ten dye-loaded cells were γEVG-responding cells, while MPG + IMP stimulation evoked responses in 8 out of 132 cells. We did not identify cells that responded to both substances.
Figure 6.
CaSR is found in distinct cells that do not express an umami/sweet receptor subunit.
(A–C) A longitudinal section of a circumvallate taste bud immunostained with antibodies against CaSR (A) and T1R3 (B). (C) Overlay of A and B. (D) A transverse section of a circumvallate taste bud immunostained with antibodies against CaSR (green) and T1R3 (red). Scale bars 20 µm.