Expression of Prostatic Acid Phosphatase in Rat Circumvallate Papillae

Abstract

ATP and its metabolites are important for taste signaling in taste buds, and thus a clearance system for them would play critical roles in maintenance of gustatory function. A previous report revealed that mRNAs for ecto-5'-nucleotidase (NT5E) and prostatic acid phosphatase (PAP) were expressed by taste cells of taste buds, and NT5E-immunoreactivity was detected in taste cells. However, there was no information on PAP-immunoreactivity in taste buds. In this study, we examined the expression profile of PAP in rat taste buds. In the isolated rat taste buds, we detected expression of mRNA for PAP, but NT5E was not detected differing from the case of mouse ones (Dando et al., 2012, J Neuroscience). On immunohistochemical analysis, PAP-immunoreactivity was found predominantly in NTPDase2-positive type I and SNAP25-positive type III taste cells, while there were no apparent signals of it in PLC-β2-positive type II, α-gustducin-positive type II, AADC-positive type III and 5HT-positive type III ones. As for NT5E, we could not detect its immunoreactivity in rat taste buds, and co-localization of it with any taste cell markers, although mouse taste buds expressed NT5E as reported previously. These findings suggest that PAP expressed by type I and one of type III taste cells of rats may contribute to metabolic regulation of the extracellular levels of adenine nucleotides in the taste buds of circumvallate papillae, and the regulating mechanisms for adenine nucleotides in taste buds might be different between rats and mice.

Fig 1. mRNA expression of PAP and NT5E in rat CP.

Taste buds were isolated from digested epithelial tissues of rat CP by means of a patch pipette (A). Expression of Pap and Nt5e was examined by nested PCR (B). Ntpdase2, Plcb2, Gust, Aadc and Kcnq1 served as positive controls for taste buds including taste cells. Taste buds, CP and N.C. denotes taste buds that were isolated with a patch pipette (arrowhead, A), taste bud-containing epithelium in rat CP and negative control (H₂O), respectively.

Fig 2. Immunohistochemical localization of PAP and NT5E in transverse and longitudinal sections of rat CP.

Representative photomicrographs for double staining with PAP (green) and NT5E (red) are shown for transverse (A) and longitudinal (B) sections. The nuclei were counterstained with Hoechst 33258 (blue). In the insets, cryosections were treated with the first antibody-free solution. Immunoreactivity due to the second antibodies only was used as a negative control, as shown with Alexa Fluor^® 594 or fluorescein isothiocyanate (FITC). Scale bar, 50 μm.

Fig 3. Immunohistochemical localization of PAP in transverse sections of rat CP.

Representative photomicrographs for double staining with PAP (green), and the type I cell marker NTPDase2 (A, red), the type II cell marker PLC-β2 (B, red), or α-gustducin (C, red), or the type III cell marker SNAP25 (D, red), AADC (E, red), or 5HT (F, red) are shown for transverse sections. In the photomicrographs on the right, cryosections were treated with the first antibody-free solution. Immunoreactivity due to the second antibodies only was used as a negative control (N.C.). Arrowheads indicate the colocalization of PAP and taste cell markers. The nuclei were counterstained with Hoechst 33258 (blue). Scale bar, 50 μm.

Fig 4. Immunohistochemical localization of PAP in longitudinal sections of rat CP.

Representative photomicrographs for double staining with PAP (green), and the type I cell marker NTPDase2 (A, red), the type II cell marker PLC-β2 (B, red), or α-gustducin (C; red), or the type III cell marker SNAP25 (D, red), AADC (E, red), or 5HT (F, red) are shown for longitudinal sections. Arrowheads indicate the colocalization of PAP and taste cell markers. The nuclei were counterstained with Hoechst 33258 (blue). Scale bar, 50 μm.

Fig 5. Immunohistochemical localization of NT5E in transverse sections of rat CP.

Representative photomicrographs for double staining with NT5E (A, green; B and C, red), and the type I cell marker NTPDase2 (A, red), the type II cell marker PLC-β2 (B, green), or the type III cell marker SNAP25 (C, green) are shown. In panel A, double staining for NT5E and NTPDase2 was performed directly using Alexa Fluor^®-conjugated primary antibodies, as described under Materials and Methods. The nuclei were counterstained with Hoechst 33258 (blue). In the photomicrographs on the right, cryosections were treated with the first antibody-free solution, and immunoreactivity due to the second antibodies only was used as a negative control (N.C.), as shown with Alexa Fluor^® 488 and/or 594. Scale bar, 50 μm.

Fig 6. Immunohistochemical localization of NT5E in longitudinal sections of rat and mouse CP.

Representative photomicrographs for double staining with NT5E (red) and type III cell marker SNAP25 (green) in longitudinal sections of SD rat (A) and C57BL/6 mouse (B) CP are shown. The nuclei were counterstained with Hoechst 33258 (blue). In the photomicrographs on the right, cryosections were treated with the first antibody-free solution. Immunoreactivity due to the second antibodies only was used as a negative control (N.C.). Scale bar, 50 μm.