Tetrahymena thermophila are free-living, ciliated eukaryotes. Their behavioral response to stimuli is well characterized and easily observable, since cells swim toward chemoattractants and avoid chemorepellents. Chemoattractant responses involve increased swim speed or a decreased change in swim direction, while chemorepellent signaling involves ciliary reversal, which causes the organism to jerk back and forth, swim in small circles, or spin in an attempt to get away from the repellent. Many food sources, such as proteins, are chemoattractants for these organisms, while a variety of compounds are repellents. Repellents in nature are thought to come from the secretions of predators or from ruptured organisms, which may serve as “danger” signals. Interestingly, several peptides involved in vertebrate pain signaling are chemorepellents in Tetrahymena, including substances P, ACTH, PACAP, VIP, and nociceptin. Here, we characterize the response of Tetrahymena thermophila to three different isoforms of nociceptin. We find that G-protein inhibitors and tyrosine kinase inhibitors do not affect nociceptin avoidance. However, the calcium chelator, EGTA, and the SERCA calcium ATPase inhibitor, thapsigargin, both inhibit nociceptin avoidance, implicating calcium in avoidance. This result is confirmed by electrophysiology studies which show that 50？ M nociceptin-NH2 causes a sustained depolarization of approximately 40？mV, which is eliminated by the addition of extracellular EGTA. 1. Introduction Nociceptin/orphanin FQ (hereafter referred to as nociceptin) is a peptide involved in vertebrate pain signaling. The endogenous receptor for this ligand is ORL-1/NCR [1, 2]. A number of signaling pathways have been implicated in vertebrate nociceptin signaling. A partial listing of molecules involved in this signaling cascade would include Gi/o proteins [1], neuronal nitric oxide synthase (nNOS) [3], and Erk-dependent signaling [4]. In addition, signaling through the nociceptin receptor induces a reduction in calcium influx via P/Q-type calcium channels in rat brain [5]. Tetrahymena thermophila are free-living, unicellular eukaryotes. While T. thermophila do not feel pain, they are capable of sensing chemoattractants and chemorepellents in their environment. This allows them to find food and possibly to escape predation [6]. A recent review by Csaba [7] details the response of T. thermophila to a number of chemoattractants and chemorepellents, including their response to many vertebrate hormones. Indeed, T. thermophila appear to synthesize and respond to a number of
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