Nucleoside Triphosphate Diphosphohydrolase-2 (NTPDase2) is the ecto-ATPase of Type I cells in taste buds
Abstract
The presence of one or more calcium-dependent ecto-ATPases (enzymes that hydrolyze extracellular 5′-triphosphates) in mammalian taste buds was first shown histochemically. Recent studies have established that dominant ecto-ATPases consist of enzymes now called nucleoside triphosphate diphosphohydrolases (NTPDases). Massively parallel signature sequencing (MPSS) from murine taste epithelium provided molecular evidence suggesting that NTPDase2 is the most likely member present in mouse taste papillae. Immunocytochemical and enzyme histochemical staining verified the presence of NTPDase2 associated with plasma membranes in a large number of cells within all mouse taste buds. To determine which of the three taste cell types expresses this enzyme, double label assays were performed using antisera directed against the glial glutamate/aspartate transporter, (GLAST), the transduction pathway proteins phospholipaseC β2 (PLCβ2) or the G protein subunit α-gustducin, and serotonin (5HT) as markers of type I, II or III taste cells, respectively. Analysis of the double labeled sections indicates that NTPDase2 immunoreactivity is found on cell processes that often envelop other taste cells, reminiscent of type I cells. In agreement with this observation, NTPDase2 was located to the same membrane as GLAST, indicating that this enzyme is present in type I cells. The presence of ecto-ATPase in taste buds likely reflects the importance of ATP as an intercellular signaling molecule in this system.
Taste buds, the sensory endorgans mediating taste, reside in the epithelium of the tongue, palate and larynx. In rodents, lingual taste buds occur in three types of papillae: fungiform papillae scattered over the anterior portion of the tongue, circumvallate papillae on the posterior dorsal surface, and foliate papillae along the posterior lateral surface. Taste buds also occur at the oral entrance to the nasoincisor ducts, soft palate, epiglottis and larynx.
Taste buds comprise several proliferative basal cells and 50–80 elongate epithelial cells which are heterogeneous in their morphologic and cytochemical characteristics. The differentiated taste cells in rodents are classified into three morphologic types: type I, type II, and type III as determined by cell structure, shape of the nucleus and apical processes (Murray and Murray, 1967; reviewed in Finger and Simon, 2000). These cell types usually correlate with particular histochemical features. For example, immunoreactivity for the G-protein subunit, a-gustducin, is localized to a subset of type II taste cells (Boughter et al., 1997; Yang et al., 2000b). This type of taste cell also expresses the transduction pathway proteins such as phospholipase C β2 and inositol 1,4,5-triphosphate receptor 3 (PLCβ2, IP3R3; Clapp et al., 2001) as well as the molecularly defined T1R (Hoon et al., 1999) and T2R taste receptors (Adler et al., 2000; Matsunami et al., 2000). In contrast, type III taste cells express none of the transduction-associated proteins but do exhibit several neuronal features including neural cell adhesion molecule (NCAM: Nelson and Finger, 1993), voltage-dependent calcium channels (Medler et al., 2003), synaptosomal-associated protein of 25kDa (SNAP-25: Yang et al., 2000a) and the ability to accumulate serotonin (Takeda et al., 1981; Yee et al., 2001). Though type I cells account for approximately half of the cells in each taste bud, this cell population is the least well characterized in terms of immunohistochemical markers but do express the glial glutamate/aspartate transporter GLAST (Lawton et al., 2000). The presence of this glial marker coupled with the observation that type I cells embrace the other cell types is strongly suggestive that these cells function like glia cells within taste buds.
The presence of calcium-dependent ecto-ATPases (enzymes that hydrolyze extracellular 5′-triphosphates such as ATP) in mammalian taste buds has long been known as demonstrated histochemically (Iwayama and Nada, 1967; Zalewski, 1968; Iwayama, 1969; Akisaka and Oda, 1977; Barry, 1992). Since these early reports, different families of these ecto-enzymes have been identified molecularly and functionally including the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family (Zimmerman, 2001). Four members of this family, NTPDases 1, 2, 3, and 8 are anchored in the plasma membrane with catalytic sites facing the extracellular space and are implicated in the control of nucleotide levels at the cell surface (Zimmerman 2001; Bigonnesse et al., 2004; Kukulski et al., 2005). Conversely, NTPDases 4–7 mainly lie in the membranes of intracellular organelles and are not clearly involved in extracellular nucleotide signaling.
Previous studies showed gustatory nucleoside hydrolase activity with several nucleoside 5′-triphosphates (ATP, ITP, and GTP), but ATP was assumed to be the most relevant substrate based on its predominant abundance in physiological extracellular fluid (Barry, 1992). This activity most likely corresponds to NTPDases 1, 2, 3 or 8 based on their location and abilities to hydrolyze ATP: NTPDase1 hydrolyzes ATP and ADP equally well; NTPDase2 strongly prefers ATP; while NTPDase3 and NTPDase8 are functional intermediates between NTPDases 1 and 2 (Kukulski et al., 2005). The histochemical profile of taste bud ecto-ATPase reveals a strong preference for ATP over ADP (Iwayama, 1969), indicating that NTPDase2 is the most likely candidate.
The present study is aimed at molecularly identifying the predominant ecto-ATPase in taste buds, and characterizing which type of taste cell expresses this enzyme. We find that only NTPDase2 is highly abundant in taste buds and is associated with the plasma membrane of type I taste cells.