Purinergic Signalling (2008) 4:171–181 177 Fig. 4 The phospholipase C inhibitor 1-(6-(17"-3-methoxyestra-1,3,5 (10)-trien-17-yl) amino)hexyl)-1H-pyrrole-2,5-dione (U73122) blocks avoidance to 100 μM guanosine-5′-O-(3-thio)triphosphate (GTP-+-S) in Tetrahymena. Baseline avoidance was reached at a U73122 concen- tration of 1 μM. The inhibition constant IC50 of this compound was near 0.001 μM. Data shown is mean+/−standard deviation (SD) of three or more trials signals through a tyrosine kinase (Fig. 2, 3). The GTP signaling pathway also involves phospholipase C (Fig. 4), intracellular calcium (Fig. 5), NOS (Fig. 6), and guanylyl cyclase (Fig. 7). The proposed GTP signaling pathway is outlined in Fig. 8. GTP is the first chemorepellent we have studied that does not signal through a G-protein-linked receptor, unlike ATP [13] and PACAP [18, 21]. In contrast to other repellents, GTP signaling is unaffected by either GDP-"-S or pertussis toxin (unpublished data). GTP is also the only one of these three repellents whose signaling is eliminated by genistein (Fig. 2; see “Results”). Previously published cross-adaptation data [2] indicated that GTP and ATP signal through different receptors. These data are consistent with the previously published literature and add the observation that both receptors and second-messenger pathways for these two repellents appear to be different. Multiple target proteins are phosphorylated in response to GTP exposure (Fig. 3d), yet the proteins remain unidentified except for molecular weight. Phosphotyrosine immunolocalization does not help us identify these pro- teins; however, the ciliary staining (Fig. 3b) opens up the possibility of direct interaction between tyrosine kinase activity and ciliary reversal. This is worthy of note, as Christensen et al. [22] report a similar increase in ciliary phosphotyrosine labeling after exposure to the chemo- attractant insulin. However, in their report, the main protein that was phosphorylated was a 66 kDa protein, which they discovered was homologous to the insulin receptor. In the case of GTP exposure, we saw no increase in labeling of the 66 kDa protein (Fig. 3d). Taken together, these data raise the possibility that there are multiple tyrosine kinase pathways that act on cilia to increase forward swim speed in the case of a chemoattractant, or to cause ciliary reversal in the case of a chemorepellent. Becausxe there were multiple phosphotyrosine bands in our Western blot (Fig. 3d) and ciliary as well as cytosolic staining in our immunofluorescence (Fig. 3b), we see that GTP exposure has both ciliary and cytosolic effects. Ciliary reversal is seen immediately upon GTP exposure; however, other effects of GTP exposure, such as mitosis, are seen hours after GTP exposure [4]. Perhaps the cytosolic target proteins are involved in other, longer-term effects of GTP, such as mitosis. Fig. 5 Intracellular calcium is required for guanosine 5'-triphosphate (GTP) avoidance in Tetrahymena. a The membrane-permeable calcium chelator 1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetra- acetic acid, tetra (acetoxymethyl) ester (BAPTA-AM) blocks avoid- ance to 100 μM GTP-+-S in Tetrahymena. Baseline avoidance was reached at a BAPTA-AM concentration of 5 μg/μl. The inhibition constant IC50 of this compound was near 0.5 μg/μl. Data shown is mean +/− standard deviation (SD) of three trials. b The endothelial reticulum (ER) calcium adenosine triphosphatase (ATPase) inhibitor thapsigargin also blocks avoidance to 100 μM guanosine-5′-O-(3-thio) triphosphate (GTP-+-S) in Tetrahymena.. Baseline avoidance was reached at a thapsigargin concentration of 1,000 nM. The IC50 of this compound was near 1 nM. Data shown is mean +/− standard deviation (SD) of three or more trials