178 Purinergic Signalling (2008) 4:171–181 Fig. 6 Nitric oxide synthase (NOS) activity is required for guanosine 5'-triphosphate (GTP) avoidance in Tetrahymena. a The NOS inhibitor 1400W blocks avoidance to 100 μM guanosine-5′-O-(3-thio)triphos- phate (GTP-+-S) in Tetrahymena. Baseline avoidance was reached at a 1400W concentration of 100 nM. The inhibition constant IC50 of this compound was near 0.1 nM. Data shown is mean +/− standard deviation (SD) of three trials. b The NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) also blocks avoidance to 100 μM GTP-+-S in Tetrahy- mena. Baseline avoidance was reached at an L-NMMA concentration of 10 μM. The inhibition constant IC50 of this compound was near 3 μM. Data shown is mean +/− standard deviation (SD) of at least three trials Many tyrosine kinase pathways involve phospholipase C. In various Tetrahymena species, evidence for phospholi- pase C involvement has been seen in stomatin-induced differentiation [24] and in commitment to cell survival or death [25]. Studies by Leondaritis and Galanopoulou [26] suggest that Tetrahymena possess a functional phosphati- dylinositol signaling system that is similar to higher eukaryotes. Indeed, in our previous studies of PACAP [10] and ATP [13], phospholipase C involvement is also implicated. In our current study, we found that U73122 effectively eliminated GTP avoidance (Fig. 4), indicating that phospholipase C signaling is involved in behavioral avoidance. However, our calphostin C data (see “Results”) indicate that PKC, a common downstream target of phospholipase C, is not required for GTP avoidance. IP3, a second messenger generated by phospholipase C, is likely involved in GTP signaling. Although we could not test IP3 involvement directly using our behavioral assay, the fact that the ER calcium ATPase inhibitor thapsigargin elimi- nated GTP avoidance (Fig. 5b) is indirect evidence that IP3 may be binding to the ER and causing release of calcium from internal stores. Calcium is a ubiquitous signaling molecule and has been implicated in ciliary reversal in Tetrahymena. It was no surprise, then, that BAPTA-AM eliminated GTP avoidance Fig. 7 Guanylyl cyclase activity is required for guanosine 5'- triphosphate (GTP) avoidance in Tetrahymena. a 4H-8-bromo-1,2,4- oxadiazolo(3,4-d) benz(b)(1,4)oxazin-1-one (NS2028), an inhibitor of guanylyl cyclase, blocked avoidance to 100 μM guanosine-5′-O-(3- thio)triphosphate (GTP-+-S) in Tetrahymena. Baseline avoidance was obtained at 10 nM. The inhibition constant IC50 of NS2028 was approximately 0.05 nM. Data shown is mean + standard deviation (SD) of three trials. b Rp-8-Bromoguanosine-3′,5′-cyclic mono- phosphorothioate (Rp-8-Br-cGMPs), a competitive inhibitor of gua- nosine 3′3;5′monophosphate (cGMP), also blocked avoidance to 100 μM GTP-+-S in Tetrahymena. Baseline avoidance was obtained at 50 μM. The IC50 of Rp-8-Br-cGMPs was approximately 7 μM. Data shown is mean + standard deviation (SD) of three or more trials