Nucleoside triphosphate diphosphohydrolases 1, 2, 3 and 8 (NTPDases 1, 2, 3 and 8) are the dominant ectonucleotidases and thereby expected to play important roles in nucleotide signaling. Distinct biochemical characteristics of individual NTPDases should allow them to regulate P2 receptor activation differentially. Therefore, the biochemical and kinetic properties of these enzymes were compared. NTPDases 1, 2, 3 and 8 efficiently hydrolyzed ATP and UTP with Km values in the micromolar range, indicating that they should terminate the effects exerted by these nucleotide agonists at P2X1- and P2Y2,4,11 receptors. Since NTPDase1 does not allow accumulation of ADP, it should terminate the activation of P2Y1,12,13 receptors far more efficiently than the other NTPDases. In contrast, NTPDases 2, 3 and 8 are expected to promote the activation of ADP specific receptors, because in the presence of ATP they produce a sustained (NTPDase2) or transient (NTPDases 3 and 8) accumulation of ADP. Interestingly, all plasma membrane NTPDases dephosphorylate UTP with a significant accumulation of UDP, favoring P2Y6 receptor activation. NTPDases differ in divalent cation and pH dependence, although all are active in the pH range of 7.0-.5. Various NTPDases may also distinctly affect formation of extracellular adenosine and therefore adenosine receptor-mediated responses, since they generate different amounts of the substrate (AMP) and inhibitor (ADP) of ecto-5-nucleotidase, the rate limiting enzyme in the production of adenosine. Taken together, these data indicate that plasma membrane NTPDases hydrolyze nucleotides in a distinctive manner and may therefore differentially regulate P2 and adenosine receptor signaling.

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. KeywordsGustatory; ecto-ATPase; NTPDase2; taste cells; ATP signaling; mouse; taste bud 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, 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 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 hav...

Background and purpose: ARL 67156, 6-N,N-Diethyl-D-b-g-dibromomethylene adenosine triphosphate, originally named FPL 67156, is the only commercially available inhibitor of ecto-ATPases. Since the first report on this molecule, various ectonucleotidases responsible for the hydrolysis of ATP at the cell surface have been cloned and characterized. In this work, we identified the ectonucleotidases inhibited by ARL 67156. Experimental approach: The effect of ARL 67156 on recombinant NTPDase1, 2, 3 & 8 (mouse and human), NPP1, NPP3 and ecto-5 0 -nucleotidase (human) have been evaluated. The inhibition of the activity of NTPDases (using the following substrates: ATP, ADP, UTP), NPPs (pnp-TMP, Ap 3 A) and ecto-5 0 -nucleotidase (AMP) was measured by colorimetric or HPLC assays. Key results: ARL 67156 was a weak competitive inhibitor of human NTPDase1, NTPDase3 and NPP1 with K i of 1173, 1874 and 1273 mM, respectively. At concentrations used in the literature (50-100 mM), ARL 67156 partially but significantly inhibited the mouse and human forms of these enzymes. NTPDase2, NTPDase8, NPP3 and ecto-5 0 -nucleotidase activities were less affected. Importantly, ARL 67156 was not hydrolysed by either human NTPDase1, 2, 3, 8, NPP1 or NPP3. Conclusions and implications:In cell environments where NTPDase1, NTPDase3, NPP1 or mouse NTPDase8 are present, ARL 67156 would prolong the effect of endogenously released ATP on P2 receptors. However, it does not block any ectonucleotidases efficiently when high concentrations of substrates are present, such as in biochemical, pharmacological or P2X 7 assays. In addition, ARL 67156 is not an effective inhibitor of NTPDase2, human NTPDase8, NPP3 and ecto-5 0 -nucleotidase.

Innate immune responses play a critical role in controlling acute infections due to Pseudomonas aeruginosa in both mice and in humans. In this review we focus on innate immune recognition and clearance mechanisms that are important for controlling P. aeruginosa in the mammalian lung, with particular attention to those that influence the outcome of in vivo infection in murine models.