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Previous studies have identified many cDNA species that encode a variety of G protein alpha-subunits occurring in taste buds. These include the cDNA encoding a taste-bud-specific Galpha, gustducin (G(gust)). Here we carried out comprehensive analyses of Galpha species that occur in the taste buds of rat circumvallate papillae and also in their single cells isolated from the taste buds. Reverse transcriptase-polymerase chain reaction showed the presence of 10 kinds of Galpha cDNAs, including a splice variant of Galphas, among which G(gust), Galphas, Galphai2 and Galphai3 cDNAs were shown to be major species. In situ hybridization and immunohistochemistry showed that Galphai2, as well as G(gust), expressed in a subset of taste bud cells, and the frequency of Galphai2-expression appears to be higher than that of G(gust). Southern analyses of the amplified cDNA from single cells showed that each taste bud cell expresses multiple Galpha mRNA species. For example, some Galphai2-positive cells also express one or more other Galpha species, including Galphas, Galphai3 and G(gust), and there is no apparent correlation in expression among the three Galpha species.

We analyzed the differentiation of taste bud cells, by precisely describing expression profiles of cytokeratins (CKs) 8 and 14 in relation to those of marker molecules including label of 5-bromo-2'-deoxy uridine (BrdU) injected. In rat circumvallate papillae, cell division was observed at the basal layer of the epithelium expressing CK14 and located outside taste buds. The progenitor cells began to migrate toward the apical surface and maintained CK14 expression at 1 day after BrdU injection (day 1). On the other hand, a minor population of newly divided cells was infrequently incorporated into taste buds and also maintained CK14 expression at day 1. In taste buds, the conversion of CK subtypes occurred from CK14 to cytokeratin 8 (CK8) at day 2-3, showing the differentiation from immature cells expressing CK14 into mature or maturing cells expressing CK8. Functionally matured cells such as taste receptor cells expressing inositol triphospate receptor type 3 (IP(3)R3) never expressed CK14, suggesting that CK14 would be expressed only in immature cells. On the other hand, a small but distinct population of BrdU-positive cells still showed CK14 immunoreactivity in taste buds even at day 12, which might correspond to the cells that remain undifferentiated for a long period within taste buds.

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