Characterization of the expression pattern of adrenergic receptors in rat taste buds

Abstract

Taste buds signal the presence of chemical stimuli in the oral cavity to the central nervous system using both early transduction mechanisms, which allow single cells to be depolarized via receptor-mediated signaling pathways, and late transduction mechanisms, which involve extensive cell-to-cell communication among the cells in the bud. The latter mechanisms, which involve a large number of neurotransmitters and neuropeptides, are less well understood. Among neurotransmitters, multiple lines of evidence suggest that norepinephrine plays a yet unknown role in the taste bud. This study investigated the expression pattern of adrenergic receptors in the rat posterior taste bud. Expression of alpha1A, alpha1B, alpha1D, alpha2A, alpha2B, alpha2C, beta1, and the beta2 adrenoceptor subtypes was observed in taste buds using RT-PCR and immunocytochemical techniques. Taste buds also expressed the biosynthetic enzyme for norepinephrine, dopamine beta-hydroxylase (DbetaH), as well as the norepinephrine transporter. Further, expression of the epinephrine synthetic enzyme, phenylethanolamine N-methyltransferase (PNMT), was observed suggesting a possible role for this transmitter in the bud. Phenotyping adrenoceptor expression patterns with double labeling experiments to gustducin, synaptosomal-associated protein 25 (SNAP-25), and neural cell adhesion molecule (NCAM) suggests they are prominently expressed in subsets of cells known to express taste receptor molecules but segregated from cells known to have synapses with the afferent nerve fiber. Alpha and beta adrenoceptors co-express with one another in unique patterns as observed with immunocytochemistry and single cell reverse transcription polymerase chain reaction (RT-PCR). These data suggest that single cells express multiple adrenergic receptors and that adrenergic signaling may be particularly important in bitter, sweet, and umami taste qualities. In summary, adrenergic signaling in the taste bud occurs through complex pathways that include presynaptic and postsynaptic receptors and likely play modulatory roles in processing of gustatory information similar to other peripheral sensory systems such as the retina, cochlea, and olfactory bulb.

Fig. 1

RT-PCR examination of adrenergic receptor expression in taste buds. RT-PCR was performed on cDNA derived from isolated whole taste buds. PCR reactions to all eight tested adrenoceptor subtypes resulted in product of appropriate size. Markers are in left lane of each gel picture.

Fig. 2

Photomicrographs of immunopositive taste receptor cells to eight expressed adrenoceptor subtypes. Single label fluorescent immunocytochemistry was performed using antibodies specific to varying adrenergic receptor subtypes on tissue samples from foliate or circumvallate papillae. Scale bar in lower right is 20 microns and applies to all examples.

Fig. 3

Examination of adrenergic related signaling molecules. Using RT-PCR of cDNA derived from whole taste buds, bands of proper size were observed for reaction with primers for DβH, NET, PNMT, β-arrestin1 and β-arrestin 2. The expected product size for each reaction is listed. Immunopositive taste receptor cells are illustrated using an antibody directed against DβH or PNMT. Scale bar in photomicrographs is 20 microns.

Fig. 4

Examination of co-expression of adrenergic receptors with gustducin. Representative photomicrographs of double label fluorescent immunocytochemistry with antibodies directed against alpha-gustducin (middle panels) and one of eight tested adrenoceptor subtypes (left panels) are illustrated. Overlaid images are at right of each panel. The scale bar in all photomicrographs represents twenty microns.

Fig. 5

Examination of co-expression of adrenergic receptors with SNAP-25. Representative photomicrographs of double label fluorescent immunocytochemistry with antibodies directed against SNAP-25 (middle panel) and one of five tested adrenoceptor subtypes (left panel) are illustrated. Overlaid images are at right of each panel. The scale bar in all photomicrographs represents twenty microns.

Fig. 6

Examination of co-expression of adrenergic receptors with NCAM. Representative photomicrographs of double label fluorescent immunocytochemistry with antibodies directed against NCAM (middle panel) and one of five tested adrenoceptor subtypes (left panel) are illustrated. Overlaid images are at right of each panel. The scale bar in all photomicrographs represents twenty microns.

Fig. 7

Immunocytochemical double labeling combinations of alpha and beta receptors. Each triplet of photomicrographs is arranged as the alpha receptor image, beta receptor image, and overlay (left to right). All scale bars represent 20 microns.

Fig. 8

Gel electrophoresis illustrating single cell RT-PCR products from eight taste receptor cells tested with a variety of primers sets to adrenoceptors or taste-related genes. Each row represents PCR results from a single primer set; each column represents results from a single cell.

Fig. 9

Qualitative Venn diagram summarizing the co-expression patterns of adrenergic receptor subtypes observed in rat posterior taste receptor cells with two additional gustatory phenotypic markers.