172 membrane [2]. The increase in Ca2+ concentration causes the cilia to reverse their direction [1], resulting in a disruption of normal swimming. This “avoidance reaction” [1] is marked by jerky movements, backward swimming, or spinning. This chemokinetic response involves a series of avoidance reactions, resulting in a “biased random walk,” which orients the organism away from the source of the repellent [3]. Avoidance to chemorepellents may be quantified by observing the response of single cells exposed to repellent using a dissection microscope. T. thermophila avoid micromolar concentrations of GTP [1]. An in vivo binding assay using [32P] GTP showed the KD for GTP binding to intact T. thermophila is 21±2.5 nM. GTP binds to its receptor with high affinity, and binding is saturable as well as reversible [1], all of which are cha- racteristic of membrane receptors. Fluorescent staining with 2′-(or 3′)-O-(2,4,6-trinitrophenyl) (TNP)-GTP has shown GTP binding on the cilia and cell surface of T. thermophila [4]. However, the second messenger pathways involved in GTP signaling have not been previously described. Recently, Iwamoto and Nakaoka [4] found that addition of extracellular GTP to the medium induces cell division in T. thermophila, a result consistent with a tyrosine kinase signaling mechanism, as tyrosine kinases are involved in many major mitotic pathways. Tyrosine phosphorylation has previously been shown to modulate the interaction of calmodulin with target proteins, including protein phospha- tase 2B (calcineurin) and neuronal nitric oxide synthase (nNOS) in an in vitro assay [5]. Tyrosine kinase activity has also been shown to activate phospholipase C, NOS (via protein kinase C), and guanylyl cyclase in the rat ileum [6]. Tyrosine kinases are frequently associated with activation of phospholipase C-+, and with release of calcium from internal stores via the IP3 pathway [7]. In our current study, we examined the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila. Our data confirm this hypothesis. In addition to tyrosine kinase signaling, we found that phospholipase C, intracellular calcium stores, NOS, and guanylyl cyclase are necessary for behavioral avoidance to occur. All of these second messengers are consistent with the existence of a tyrosine kinase signaling pathway in T. thermophila. Materials and methods Cell cultures T. thermophila, strain B2086, a generous gift from T.M. Hennessey (SUNY-Buffalo) were used for all of the experiments. Cells were grown at 25°C in the axenic medium of Dentler [8] without shaking. Two-day-old cell cultures were used for all assays described below. Purinergic Signalling (2008) 4:171–181 Chemicals and solutions Behavioral assays were carried out in a buffer of pH 7.0 containing 10 mM Trizma base, 0.5 mM MOPS, and 50 μM CaCl2. All repellents and inhibitors used were dissolved in this buffer. Compounds that were insoluble in aqueous solutions were first dissolved in a small quantity of dimethylsulfoxide (DMSO) and then diluted 1:1,000 or more into the buffer and vortexed vigorously. The chemo- repellent guanosine-5′-O-(3-thio)triphosphate (GTP-+-S) was obtained from Alexis Biochemicals (San Diego, CA, USA), along with the G-protein inhibitor pertussis toxin. The tyrosine kinase inhibitor genistein was obtained from Acros Organics (New Jersey, USA). Calphostin C [a protein kinase C (PKC) inhibitor], diadzein a control for genistein inhibition), guanosine-5′-O- (2-thiodiphosphate) (GDP-"-S) (a G-protein inhibitor), Rp-8-Bromoguanosine- 3′,5′-cyclic monophosphorothioate (Rp-8-Br-cGMPs) (a competitive inhibitor of PKG), 1-(6-(17"-3-methoxyestra- 1,3,5(10)-trien-17-yl) amino)hexyl)-1H-pyrrole-2,5-dione (U73122) (a phospholipase C inhibitor), NG-monomethyl- L-arginine (L-NMMA), and N-(3-(Aminomethyl)benzyl) acetamidine (1400W) (NOS inhibitors), 4H-8-bromo-1,2,4- oxadiazolo(3,4-d) benz(b)(1,4)oxazin-1-one (NS2028) (a PKG inhibitor), and the Quantizyme nitric oxide (NO) assay kit were purchased from BIOMOL Research Labo- ratories (Plymouth Meeting, PA, USA). The polyclonal antiphosphotyrosine antibody was purchased from Upstate (Lake Placid, NY, USA); 30X Tris-buffered saline (TBS) and 30X TBS/nonidet P40 (NP40) were obtained from Modern Biology (West Lafayette, IN, USA). The guanosine 3′3;5′monophosphate (cGMP) emzyme immunosorbent assay (EIA) kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). Sodium metavanadate (a phos- phatase inhibitor) and theophylline (a phosphodiesterase inhibitor) were obtained from Sigma Chemical (St. Louis, MO, USA). All other chemicals were purchased from Sigma as well. Behavioral assays In vivo behavioral assays were carried out as previously described [1, 9, 10]. Briefly, cells were washed in buffer, and 300 μl of cell suspension was transferred to the first well of a three-well spot microtiter plate. Cells were then individually transferred by micropipette to the second well, which contained 300 μl of the test concentration of inhibitor dissolved in buffer. After at least 10–15 min of incubation time, individual cells were transferred to the third well, which contained 300 μl GTP-+-S and the test concentration of inhibitor. Each cell was briefly observed (1–5 s) for signs of avoidance. Avoidance behavior was characterized by jerky, backward movements; swimming in