Purinergic Signalling (2008) 4:171–181 DOI 10.1007/s11302-007-9052-4 ORIGINAL PAPER GTP avoidance in Tetrahymena thermophila requires tyrosine kinase activity, intracellular calcium, NOS, and guanylyl cyclase Janine Bartholomew & Johnathan Reichart & Romie Mundy & Jacquelyn Recktenwald & Shannon Keyser & Mark Riddle & Heather Kuruvilla Received: 25 September 2006 / Accepted: 2 February 2007 / Published online: 24 February 2007 # Springer Science + Business Media B.V. 2007 Abstract Guanosine 5'-triphosphate (GTP) is a chemo- repellent in Tetrahymena thermophila that has been shown to stimulate cell division as well as ciliary reversal. Previous studies have proposed that GTP avoidance is linked to a receptor-mediated, calcium-based depolarization. However, the intracellular mechanisms involved in GTP avoidance have not been previously documented. In this study, we examine the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila. Using behavioral assays, enzyme immunosorbent assays, Western blotting, and immunofluorescence, we present data that implicate a tyrosine kinase, phospholipase C, intracellular calcium, nitric oxide synthase (NOS) and guanylyl cyclase in GTP signaling. The tyrosine kinase inhibitor genistein eliminates GTP avoidance in Tetrahymena in behavioral assays. Similarly, pharmacological inhibitors of phospholipase C, NOS, and guanylyl cyclase all eliminated Tetrahymena avoidance to GTP. Immunofluorescence data shows evidence of tyrosine kinase activity in the cilia, suggesting that this enzyme activity could be directly involved in ciliary reversal. Keywords Chemorepellent . Extracellular nucleotide . Purine metabolism Abbreviations EIA Enzyme immunosorbent assay cGMP Guanosine 3′3;5′monophosphate GDP-"-S Guanosine-5′-O- (2-thio)diphosphate J. Bartholomew : J. Reichart : R. Mundy : J. Recktenwald : S. Keyser : M. Riddle : H. Kuruvilla (*) Department of Science and Mathematics, Cedarville University, Cedarville, OH 45314, USA e-mail: heatherkuruvilla@cedarville.edu GTP-+-S PKC NO NOS Rp-8-Br- cGMPs TNP-GTP Introduction Guanosine-5′-O-(3-thio)triphosphate Protein kinase C Nitric oxide Nitric oxide synthase Rp-8-Bromoguanosine-3′,5′-cyclic monophosphorothioate 2′-(or 3′)-O-(2,4,6-trinitrophenyl)guanosine 5′-triphosphate Chemorepellents are molecules that inhibit or repel the migration of individual cells. In protozoans, chemorepel- lents are hypothesized to serve a defense mechanism, helping the organism to avoid predation and environmental hazards. For example, a damaged Tetrahymena thermophila cell may leak cytosolic purines such as adenosine triphos- phate (ATP) or guanosine 5'-triphosphate (GTP) into the surrounding environment, causing other cells to avoid the area, thereby avoiding the source of damage to the original cell and increasing the survival rate of the species [1, 2]. It is possible that cytosolic purines may be secreted by these organisms as a means of communication between cells (analogous to paracrine secretion) or in order to trigger second-messenger pathways within the cell doing the secretion (analogous to autocrine secretion). At this time, secretion of purine nucleotide by Tetrahymena has not been documented, although Tetrahymena have been shown to secrete a number of enzymes and peptides. In T. thermophila, extracellular GTP is a known chemo- repellent [1, 3, 4]. GTP causes depolarization in the somatic membrane [2], which has been proposed to trigger opening of the voltage-dependent calcium channels in the ciliary