Heather G. Kuruvilla

Biochemical Evidence for a P2Y-like Receptor in Tetrahymena thermophila

Extracellular nucleotides are ubiquitous signaling molecules. ATP signals through two receptor types: the ionotropic P2X receptors, and the metabotropic P2Y receptors. ATP acts as a chemorepellent in Tetrahymena thermophila, where it causes a distinct avoidance response. The intracellular mechanisms by which ATP causes avoidance in this organism, however, are unknown. In this study, we use in vivo pharmacological assays along with enzyme immuno-assays to obtain information about the ATP chemorepellent pathway and its associated second messenger systems. Our data show strong similarities between the presumed ATP receptor of T. thermophila and members of the P2Y family of receptors. The ATP response of T. thermophila appears to be coupled to phospholipase C, a defining characteristic of the P2Y receptor family. In addition, the ATP chemoresponse appears to be linked to a Gi/o protein, nitric oxide synthase, and adenylyl cyclase, all of which are characteristic of some P2Y receptors. This is an important first step in describing the pathways involved in ATP chemoresponse of this organism.

PACAP-38 is a chemorepellent and an agonist for the lysozyme receptor in tetrahymena thermophila.

Abstract Pituitary adenylate cyclase activating peptide (PACAP-38) is a peptide hormone which functions in many mammalian systems, including the nervous and digestive systems. Using in vivo behavioral studies, we have found that this hormone functions as a chemorepellent in Tetrahymena thermophila with an EC50 of 10 nM. Cells previously adapted to PACAP-38 were found to be adapted to lysozyme, and vice versa. Furthermore, the in vivo behavioral activity of PACAP-38 was blocked by addition of the anti-lysozyme receptor antibody, 5545. Chemorepellent activity of PACAP-38 was also inhibited by the addition of neomycin sulfate (inhibition constant Ki=0.080 μmol · l−1), a competitive inhibitor of lysozyme binding to its receptor. PACAP-38 is a more potent and specific agonist for the lysozyme receptor than either intact lysozyme or CB2, a 24-amino acid fragment of lysozyme.

Chemosensory Responses of Tetrahymena thermophila to CB2, a 24-Amino Acid Fragment of Lysozyme

Abstract While lysozyme is a depolarizing chemorepellent in Tetrahymena, the entire lysozyme molecule is not necessary to activate the lysozyme receptor. Reduced lysozyme was cut into three fragments by cyanogen bromide cleavage and the fragments (CB1, CB2 and CB3) were separated by HPLC. Behavioral bioassays showed that the carboxy-terminal 24-amino-acid fragment, which we call CB2, is 100 times more active than intact lysozyme as a chemorepellent. CB2 appears to activate the same receptor as lysozyme because behavioral cross-adaptation is seen between these two compounds and an antibody generated to the purified lysozyme receptor blocks responses to both lysozyme and CB2. This is further supported by the observation that neomycin, which is a competitive inhibitor of lysozyme binding, also inhibits CB2 responses. This inhibition may be due to the fact that neomycin is highly positively charged (+5 at pH 7.0) and CB2 has a net charge of +4 at pH 7.0. Intracellular electrophysiological recordings documented that CB2 elicits a transient, depolarizing receptor potential that is similar to the lysozyme-induced depolarizations except they are much smaller. CB2 is a more potent and specific ligand for use in studies of the lysozyme receptor of Tetrahymena.

A comparison of the polycation receptors of Paramecium tetraurelia and Tetrahymena thermophila.

ABSTRACT. Chemorepellents are compounds that cause ciliated protozoans to reorient their swimming direction. A number of chemorepellents have been studied in the ciliated protozoans, Paramecium and Tetrahymena. Chemorepellents, such as polycations, cause the organism to exhibit “avoidance behavior,” a swimming behavior characterized by jerky movements and other deviations from normal forward swimming, which result from ciliary reversal. One well‐characterized chemorepellent pathway in Tetrahymena is that of the proposed polycation receptor that is activated by lysozyme and pituitary adenylate cyclase activating polypeptide (PACAP). In this study, we compare the response of Paramecium to the chemorepellents lysozyme, vasoactive intestinal peptide (VIP), and PACAP to the previously studied polycation response in Tetrahymena. Our results indicate that lysozyme, VIP, and PACAP are all chemorepellents in Paramecium, just as they are in Tetrahymena. However, the signaling pathways involved appear to be different. While previous pharmacological characterization indicates that G‐proteins are involved in polycation signaling in Tetrahymena, we present evidence that similar reception in Paramecium involves activation of a tyrosine kinase pathway in order for lysozyme avoidance to occur. Polycation responses of both organisms are inhibited by neomycin sulfate. While PACAP is the most effective of the three chemorepellents in Tetrahymena, lysozyme is the most effective chemorepellent in Paramecium.

Nociceptin Signaling Involves a Calcium-Based Depolarization in Tetrahymena thermophila

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 μM nociceptin-NH2 causes a sustained depolarization of approximately 40 mV, which is eliminated by the addition of extracellular EGTA.

Netrin-1 Peptide Is a Chemorepellent in Tetrahymena thermophila

Netrin-1 is a highly conserved, pleiotropic signaling molecule that can serve as a neuronal chemorepellent during vertebrate development. In vertebrates, chemorepellent signaling is mediated through the tyrosine kinase, src-1, and the tyrosine phosphatase, shp-2. Tetrahymena thermophila has been used as a model system for chemorepellent signaling because its avoidance response is easily characterized under a light microscope. Our experiments showed that netrin-1 peptide is a chemorepellent in T. thermophila at micromolar concentrations. T. thermophila adapts to netrin-1 over a time course of about 10 minutes. Netrin-adapted cells still avoid GTP, PACAP-38, and nociceptin, suggesting that netrin does not use the same signaling machinery as any of these other repellents. Avoidance of netrin-1 peptide was effectively eliminated by the addition of the tyrosine kinase inhibitor, genistein, to the assay buffer; however, immunostaining using an anti-phosphotyrosine antibody showed similar fluorescence levels in control and netrin-1 exposed cells, suggesting that tyrosine phosphorylation is not required for signaling to occur. In addition, ELISA indicates that a netrin-like peptide is present in both whole cell extract and secreted protein obtained from Tetrahymena thermophila. Further study will be required in order to fully elucidate the signaling mechanism of netrin-1 peptide in this organism.