Purinergic Signalling (2008) 4:171–181 Fig. 8 Current model of guano- sine 5'-triphosphate (GTP) sig- naling. Based on data obtained in this experiment, GTP appears to signal through a receptor linked to a tyrosine kinase. Phospholipase C, intracellular calcium release, nitric oxide (NO) production, and guanylyl cyclase are all involved in GTP avoidance. Some of the rela- tionships between pathway components remain unknown. This model is based on the data presented in this paper, along with data obtained from the literature [26, 32, 33] 179 (Fig. 5a), as it has previously been shown to eliminate avoidance to PACAP [18] and ATP [13]. Calcium is likely to play multiple roles in signaling. One possible scenario is the direct interaction of calcium with ciliary motor proteins. For example, inner arm dynein 1 (I1) is required for calcium-dependent ciliary reversals in T. thermophila [27]. In this study, a “calcium sensor,” which interacts with I1, was postulated. This calcium sensor protein remains to be isolated. Intracellular calcium may also play other, less direct, roles in GTP avoidance. Indeed, in the protozoan parasite Leishmania donovani, both NOS and guanylyl cyclase are activated by intracellular calcium [28]. Both NOS (Fig. 6) and guanylyl cyclase (Fig. 7) activity are required for GTP avoidance. Previous studies that implicate NOS and guanylyl cyclase in Tetrahymena [13, 18, 19] do not indicate the type of NOS or guanylyl cyclases involved, though Christensen et al. [19] give evidence for a soluble guanylyl cyclase. In L. donovani, there is evidence for a constitutive, neuronal-type NOS that then activates a soluble guanylyl cyclase [28]. Both of these enzymes are activated by intracellular calcium. Our data would be consistent with a similar mechanism in Tetrahymena, as proposed in our model of GTP signaling (Fig. 8). However, neither a soluble guanylyl cyclase nor an nNOS isoform have been purified from Tetrahymena to date. Calcium-binding proteins, such as calmodulin, may also be involved in GTP signaling in Tetrahymena, either by interacting with enzymes that generate second messengers or by interacting with the molecular motors responsible for ciliary reversal. Previous studies [29, 30] indicate that calmodulin activates a ciliary guanylyl cyclase in this organism. Our studies leave open the possibility that this cyclase, activated by calcium/calmodulin, is involved in GTP signaling. In addition to this enzyme, Hirano-Ohnishi and Watanabe [31] reported a calmodulin-dependent phos- phorylation of ciliary beta tubulin in Tetrahymena, which they believe to be involved in ciliary reversal. Elongation factor 1α, which functions in protein translation, also interacts with ciliary calmodulin [32]. Whereas this would not necessarily function in ciliary reversal, it may modulate some of the longer-term effects of GTP. Finally, Ueno et al. [33] report that ciliary calmodulin interacts with proteins that are homologues of radial spoke proteins in Chlamydo- monas, further implicating calmodulin directly in ciliary reversal. We have added some of these proteins to our model of GTP signaling (Fig. 8), though there are doubtless more proteins to be discovered. In this study, we used behavioral assays and pharmaco- logical inhibitors to explore the intracellular mechanisms employed in GTP signaling in T. thermophila. Because pharmacological inhibitors often have unknown side effects, we used multiple inhibitors when available. Some of these inhibitors had to be used at higher concentrations in our study than have been previously published for mammalian cells. Perhaps this is because of the differences in the structure of the Tetrahymena proteins compared with their mammalian counterparts. Recognizing the potential