Glucose transporter/T1R3-expressing cells in rat tracheal epithelium

Abstract

Glucose transport plays an important role in maintaining low sugar concentration in airway surface liquid (ASL), which is critical for mucociliary clearance and bacterial colonization. Experimental evidence indicates that glucose/hexose uptake in lung/airway cells occurs by means of two structurally distinct glucose transporter pathways: the Na(+) -dependent glucose transporters (SGLT family) and the facilitative glucose transporters (GLUT family). In this study, we examined the expression of the major glucose transporters of the intestine, GLUT2, GLUT5, SGLT1 and T1R3 taste receptor subunit, in the trachea of rats using immunohistochemistry and immunoelectron microscopy, and compared them using double-labeled confocal microscopy. We found that GLUT2, GLUT5, SGLT1 and T1R3 are selectively expressed in different cell types. T1R3 and GLUT2 are predominantly expressed in subsets of solitary chemoreceptor cells (SCCs) and ciliated cells, GLUT5 is present in subsets of SCCs and in secretory cells, and SGLT1 is exclusively expressed in a unique cell type, SCCs. Furthermore, we demonstrated that T1R3 is colocalized with SGLT1 in SCCs and with GLUT2 transporter in ciliated cells. In conclusion, these findings reveal that different cell types are associated with the uptake of glucose in ASL and that, due to their T1R3 expression, SCCs and ciliated cells are most likely to participate in the chemosensory process in ASL.

Fig. 1

Immunoperoxidase localization of GLUT2 in rat airway showing immunoreactivity in bronchiolar (A,B), tracheal ciliated (C,D), and solitary chemosensory cells (E). No labeling was observed when the anti-GLUT2 antibody was preabsorbed with its specific peptide (F). *Basement membrane; c, cilia; SCC, solitary chemosensory cell. Scale bar: 30 μm (A), 10 μm (B–F).

Fig. 2

Immunoelectron microscopy for GLUT2 in ciliated (CC) and solitary chemosensory cells (SCC) of the trachea. In CC, GLUT2 immunoreactivity is concentrated on the apical surface (arrow, A); cilia do not show immunostaining (B). In SCC, GLUT2 immunoreactivity is distributed throughout the entire cytoplasm (arrow; C) and in the brush-like set of microvilli on the apical surface (D). ap, apical pole; c, cilia; m, microvilli; n, nucleus; * Brush-like set of microvilli. Scale bar: 500 nm (A,C,D), 100 nm (B).

Fig. 3

Immunoperoxidase staining showing GLUT5 immunoreactivity at light (A–F) and electron microscopy (G, H) in rat trachea. Immunoreactivity is present in secretory (A–E), and solitary chemosensory cells (F). Nuclei are stained with toluidine blue (A). At ultrastructural level, secretory cells show GLUT5 labeling around or inside the granules (arrow, G), or sparsely distributed in the apical surface (arrow, H). *Basement membrane; c, cilia. Scale bar, 10 μm (A–F), 2000 nm (G, H).

Fig. 4

Immunoperoxidase staining showing SGLT1 immunoreactivity in solitary chemosensory cells of the trachea. The labeling is cytoplasmic from the body to the apex of the cells. *Basement membrane. Scale bar: 5 μm.

Fig. 5

Immunoelectron microscopy showing expression of SGLT1 in solitary chemosensory cells of rat trachea. SGLT1 immunoreactive cells are pear- or flash-shaped cells with an apical process (A) with thin microvilli protruding into the lumen (B). Some cells are characterized by the brush-like set of microvilli in their apical process (D,E). Immunoreactivity is expressed throughout the entire cytoplasm (A,C,D), and in the apical microvilli (B,E). The boxed area in (D) is shown at higher magnification in (E). CC, ciliated cell; SCC, solitary chemosensory cell; ap, apical pole; m, microvilli; n, nucleus; *Brush-like set of microvilli. Scale bar: 2000 nm (A, C, D), 500 nm (B, E).

Fig. 6

Immunoperoxidase (A,D) and immunofluorescent (green; B,C) staining showing T1R3 immunoreactivity in ciliated (A–C) and solitary chemosensory cells (D) of rat trachea. Nuclei are stained with DAPI (B). Ciliated cells show immunolabeling in the apical surface beneath the cilia (A), in some spots along the cilia (B,C), and on the basolateral membrane (arrow; C). Solitary chemosensory cells show diffuse immunolabeling in the cytoplasm. *Basement membrane; c, cilia; SCC, solitary chemosensory cell. Scale bar: 15 μm.

Fig. 7

Double-immunofluorescent confocal microscopy showing expression of α-tubulin (red) with GLUT2 (green) in tracheal cells (A–F). The merge is represented in panels (C,F,I). Immunostaining for α-tubulin and GLUT2 in immunoreactive cells is never colocalized (D–F). Absence of labeling is observed when anti-GLUT2 antibody was preincubated with the control peptide (G–I). Scale bar: 30 μm (C), 20 μm (I), 10 μm (F).

Fig. 8

Double-immunofluorescent confocal microscopy showing expression of α-tubulin (red) with GLUT5 (green; A–F), or SGLT1 (green; G–L), or α-gustducin (green; M–O) in tracheal cells. The merge is represented in panels (C, F, I, L, O). GLUT5 immunoreactivity is observed in α-tubulin-negative cells identified as secretory cells (A–C) and in basal cells (D–F). SGLT1 and α-gustducin immunoreactivities are observed in α-tubulin-negative cells identified as solitary chemosensory cells (G–L and M–O, respectively). Scale bar: 30 μm (I), 20 μm (C,F,L,O).

Fig. 9

Double-immunofluorescent confocal microscopy showing expression of α-tubulin (red) with T1R3 (green) in tracheal cells. The merge is represented in panels (C,F,I). α-Tubulin and T1R3 are colocalized in some spots on the cilia (A–F). Labeling for T1R3 is also observed in the apical cytoplasm beneath the cilia, and on the basolateral membrane of ciliated cells (D–F). Double-labeling is not observed when anti-α-tubulin antibody was omitted (G–I). Scale bar: 20 μm (C), 10 μm (F,I).

Fig. 10

Double-immunofluorescent confocal microscopy showing expression of GLUT2 (red, A–F), or GLUT5 (red, G–I), or SGLT1 (red, J–O) with T1R3 (green) in tracheal cells. The merge is represented in panels (C,F,I,L,O). T1R3 and GLUT2 are colocalized beneath the apical membrane of ciliated cells (A–F). Labeling for GLUT5 is observed in solitary chemosensory cells which lack T1R3 expression (G–I). Labeling for SGLT1 is present in solitary chemosensory cells which are T1R3-positive (J–L) or T1R3-negative (M–O). Scale bar: 50 μm (O), 30 μm (C), 20 μm (F,I,L).

Fig. 11

The diagram shows a simplified summary of glucose transporters (GLUT2, GLUT5, SGLT1) and T1R3 immunolocalization in different cell types of rat tracheal epithelium. Each cell type is indicated by a specific color: ciliated cell is represented in yellow, solitary chemosensory cell in green, basal cell in red, and secretory cell in light blue. Cell nuclei are colored in gray.