176 In many cells, activation of a tyrosine kinase activates phospholipase C-+. We used the phospholipase C inhibitor, U73122, to determine whether phospholipase C might be involved in GTP avoidance. GTP avoidance was effectively eliminated at a U73122 concentration of 1 μM. This concentration was ten times lower than that used to inhibit phospholipase C in molluscan ciliary cells [12]. The IC50 of this compound was approximately 0.001 μM. Phospholi- pase C activates the PKC pathway through the generation of diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP3). PKC is then activated by DAG binding in the presence of calcium. To determine whether PKC was involved in GTP avoidance, we used the PKC inhibitor calphostin C, which competes for DAG binding to PKC. In the presence of 10 μM calphostin C, avoidance to 100 μM GTP-+-S was reduced only to 96.6 +/− 5.8%, compared with 100% avoidance in control cells. This concentration of calphostin C eliminated avoidance to 150 μM ATP-+-S [13]. Because calphostin C had no measurable effect on GTP avoidance, we concluded that PKC activy is not required for GTP signaling. It is very likely that the two second mes- sengers, IP3 and DAG, are generated by phospholipase C and that PKC is activated, but that PKC targets cellular substrates that are not directly involved in ciliary reversal. Because calcium signaling is often associated with tyrosine kinase pathways and because calcium influx is associated with GTP exposure [2], we decided to eliminate calcium rom cells using the membrane-permeable calcium chelator 1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'- tetraacetic acid, tetra (acetoxymethyl) ester (BAPTA-AM). As seen in Fig. 5a, exposure to this calcium chelator elim- inated avoidance in a concentration-dependent manner, with baseline avoidance seen at a BAPTA-AM concentra- tion of 5 μg/ml. This result was certainly not surprising, as all known chemorepellents that have been studied electro- physiologically in Tetrahymena have elicited depolariza- tions [2, 14, 15] that have been proposed to be linked to calcium. Because our data indicated that phospholipase C was involved in GTP avoidance, we decided to test the hypothesis that internal calcium stores may also contribute to avoidance, consistent with the liberation of IP3 from the plasma membrane. We used an inhibitor of the endoplasmic reticulum (ER) calcium ATPase, thapsigargin, to deplete ER calcium stores to determine whether these stores were necessary for avoidance. Baseline avoidance to GTP was seen at a thapsigargin concentration of 1,000 nM (Fig. 5b). The IC50 of thapsigargin was approximately 1 nM. NO is another second messenger sometimes associated with tyrosine kinases [5, 17] and often associated with calcium signaling [5, 18]. In addition, NO has been found to be a critical second messenger in the Tetrahymena response to both ATP [13] and PACAP-38 [18]. To determine whether NO was involved in GTP signaling, Purinergic Signalling (2008) 4:171–181 we used the NOS inhibitors 1400W (Fig. 6a) and L- NMMA (Fig. 6b). Both inhibitors blocked avoidance to 100 μM GTP-+-S in Tetrahymena, with baseline avoidance being reached at 100 nM for 1400W (Fig. 6a) and 50 μM for L-NMMA (Fig. 6b). The IC50 of 1400W was near 0.1 nM, whereas L-NMMA was approximately 3 μM. To confirm the activity of NOS in GTP avoidance, we used the Quantizyme NO assay to determine whether there was a difference in NO metabolism between control and GTP-stimulated cells. Cells stimulated with 8 mg/ml GTP had a 146+/− 4.7% increase in NO levels over control cells, consistent with the inhibitor studies (n = 3). In many cells, NO activates guanylyl cyclase, which then produces cGMP. Previous studies by Christensen et al. [20] described an NO-linked, possibly soluble guanylyl cyclase activity in T. thermophila. More recent studies, reviewed by Linder and Schultz [20], described a mem- brane-associated and calcium-dependent guanylyl cyclase in these organisms. To determine whether any guanylyl cyclase isoform was involved in GTP avoidance, we used NS2028, an inhibitor of guanylyl cyclase (Fig. 7a), as well as Rp-8-bromo-cGMPs, a competitive inhibitor of cGMP (Fig. 7b), in behavioral assays. NS2028 and Rp-8-bromo- cGMPs both blocked avoidance to 100 μM GTP-+-S in Tetrahymena. Baseline avoidance was obtained at 10 nM NS2028 (Fig. 7a) and at 50 μM Rp-8-bromo-cGMPs (Fig. 7b). The IC50 of NS2028 was approximately 0.05 nM and of Rp-8-bromo-cGMPs near 7 μM. To confirm these results biochemically, we performed an EIA assay to determine whether GTP exposure resulted in elevated cGMP levels within Tetrahymena. Exposure to 5 mg/ml GTP resulted in a 1,119 +/− 4.2% increase above control cGMP levels (n ≥3), consistent with the hypothesis that GTP avoidance is linked to a guanylyl cyclase. Based on the data we collected, we put together a provisional model of GTP signaling (Fig. 8). In our model, GTP binds to a receptor, which activates tyrosine kinase while interacting with a membrane calcium channel, causing a depolarization. Phospholipase C is activated, generating IP3, which allows calcium efflux from ER stores. The high cytosolic calcium interacts with some (unknown) isoform of NOS, guanylyl cyclase, and other calcium-binding proteins, ultimately resulting in ciliary reversal. Whereas some of the relationships between second messengers are still unknown, the model has been put together to stimulate thought and discussion and to make some order out of a complex pathway. Discussion The data we have compiled here are consistent with the hypothesis that the previously described GTP receptor [1]