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Nociceptin Signaling Involves a Calcium-Based Depolarization in Tetrahymena thermophila

Nociceptin Signaling Involves a Calcium-Based Depolarization in Tetrahymena thermophila Hindawi Publishing Corporation International Journal of Peptides Volume 2013, Article ID 573716, 7 pages http://dx.doi.org/10.1155/2013/573716 Research Article Nociceptin Signaling Involves a Calcium-Based Depolarization in Tetrahymena thermophila 1 2 2 2 Thomas Lampert, Cheryl Nugent, John Weston, Nathanael Braun, and Heather Kuruvilla Department of Biological Sciences, State University of New York at Buaff lo, 109 Cooke Hall, Buaff lo,NY14260,USA Department of Science and Mathematics, Cedarville University, 251 North Main Street, Cedarville, OH 45314, USA Correspondence should be addressed to Heather Kuruvilla; heatherkuruvilla@cedarville.edu Received 7 March 2013; Accepted 9 April 2013 Academic Editor: Hubert Vaudry Copyright © 2013 Thomas Lampert et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tetrahymena thermophila are free-living, ciliated eukaryotes. eTh ir 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 𝜇 M nociceptin-NH2 causes a sustained depolarization of approximately 40 mV, which is eliminated by the addition of extracellular EGTA. 1. Introduction their environment. This allows them to find food and possibly to escape predation [6]. A recent review by Csaba [7] details Nociceptin/orphanin FQ (hereaer ft referred to as nociceptin) the response of T. thermophila to a number of chemoat- is a peptide involved in vertebrate pain signaling. The tractants and chemorepellents, including their response to endogenous receptor for this ligand is ORL-1/NCR [1, 2]. many vertebrate hormones. Indeed, T. thermophila appear A number of signaling pathways have been implicated in to synthesize and respond to a number of vertebrate hor- vertebrate nociceptin signaling. A partial listing of molecules mones, including serotonin, melatonin, adrenocorticotropic involved in this signaling cascade would include G proteins 𝑖/𝑜 hormone, and insulin [7]. [1], neuronal nitric oxide synthase (nNOS) [3], and Erk- A number of chemorepellents which have been charac- dependent signaling [4]. In addition, signaling through the terized in T. thermophila are polycationic peptides, including nociceptin receptor induces a reduction in calcium influx via lysozyme [8], thelysozymefragmentCB2 [9], PACAP [10], P/Q-type calciumchannelsinrat brain[5]. and nociceptive peptides including bradykinin and substance Tetrahymena thermophila are free-living, unicellular P[11]. Lysozyme signaling involves a calcium-based depolar- eukaryotes. While T. thermophila do not feel pain, they are ization [12]. Lysozyme and PACAP appear to share a signaling capable of sensing chemoattractants and chemorepellents in pathway [9], which involves cAMP and phospholipase C 2 International Journal of Peptides [13], as well as NOS and cGMP [14]. A related peptide, Cross-adaptation assays were performed as previously VIP, also uses these signaling pathways and cross-adapts described [10, 17]. Briefly, 300 𝜇 Lofcells were placed into with lysozyme and PACAP, suggesting that Tetrahymena are the rfi st well of a 3-well microtiter plate. Cells were then signaling through a generalized polycation receptor [15]. individually transferred to the second well of the 3-well Nociceptin is a polycationic peptide that is commercially microtiter plate, which contained a repellent. eTh cells were available in three different isoforms. Nociceptin carries a allowed to adapt to this repellent for 10–15 minutes or until charge of +4 at pH 7.0, while nociceptin-NH2 carries a charge cells showed baseline avoidance (an avoidance of no more of +5 at pH 7.0. Nociceptin-Arg Lys carries a charge of +6 than 20%). Cells were then individually transferred to the 14 15 at pH 7.0. Our hypothesis was that all three of the nociceptin third well of the 3-well microtiter plate, which contained the analogues would be chemorepellents in T. thermophila and repellent to be tested for cross-adaptation and monitored for that more highly charged nociceptin isoforms will have a avoidance behavior. Data which showed 20% or fewer cells lower EC in behavioral assays than isoforms which carry exhibiting avoidance was considered “baseline avoidance.” alessercharge. Baseline avoidance is the number of cells in our assay which show avoidance behavior when being transferred from one well containing buffer to another well containing the same 2. Materials and Methods buffer and usually ranges from 5 to 20%. Cells exhibiting baseline avoidance in response to this assay were considered to be cross-adapted. Each trial represents 10 cells. A minimum 2.1. Cell Cultures. Tetrahymena thermophila,strainB2086,a of 6 trials was performed for each data point. generous gift from Hennessey [ 6] (SUNY Buffalo), was used for all of the experiments. Cells were grown at 25 Cinthe axenic medium of Dentler [16], without shaking or addition 2.4. Electrophysiology. Standard one-electrode whole-cell of antibiotics. Two-day old cell cultures were used for all membrane potential recordings were recorded as the pre- behavioral assays described below. viously reported procedures in Tetrahymena thermophila [9, 12]. eTh recording bueff r contained were carried out in a buffer of pH 7.0 containing 10 mM Trizma base, 2.2. Chemicals and Solutions. Behavioral assays were car- 0.5 mM MOPS, and 1 mM CaCl . Membrane potentials ried out in a buffer of pH 7.0 containing 10 mM Trizma were displayed on a digital oscilloscope and retained on base,0.5mM MOPS,and 50𝜇 M CaCl . All repellents and a chart recorder during continuous bath perfusion at a inhibitors used were dissolved in this bueff r. rate of approximately 20.0 mL/min. The recording bath had All nociceptin isoforms, thapsigargin, J-113397, and a volume of approximately 1 mL. Solutions were changed EGTA, were purchased from Tocris Biosciences, Bristol, UK. by switching valves connected either to bueff r or to the experimental solution without changing the flow rate of the 2.3. Behavioral Assays. Behavioral assays were carried out perfusion system. as previously described [8, 10, 17]. Ten milliliters of T. thermophila culture was washed by centrifugation in a clinical centrifuge at high speed, and the pellet was reconstituted in 10 mL bueff r. This wash step was repeated twice, and cells were 3. Results reconstituted in 5 mL of buffer for use in behavioral assays. To perform the behavioral assays, 300𝜇 Lofcellsuspensionwas All isoforms of nociceptin were chemorepellents in T. ther- transferred to the rfi st well of a microtiter plate. Cells were mophila (Figure 1). Nociceptin, which has a charge of +4 at then transferred individually using a micropipette into the our assay pH of 7.0, had an EC of 100𝜇 Minour behavioral second well of the microtiter plate, which contained 300𝜇 L assay. Nociceptin-NH2, which has a charge of +5 under assay of buffer as a control. Cells were then transferred to a third conditions, had an EC of 50𝜇 M, while nociceptin-Arg - 100 14 well containing 300𝜇 L of nociceptin. Behavior of the cells was Lys which has a charge of +6 under assay conditions had observed for the rfi st 5 seconds aeft r transfer to the third well, an EC of 25𝜇 M. Avoidance was observed for 1–5 seconds and the percentage of cells exhibiting avoidance behavior was but was seen for as long as 10–15 minutes (not shown). Aeft r noted. Varying concentrations of each peptide were used until cells acclimated to the nociceptin, they returned to forward we determined the minimum concentration at which 90% swimming. of the cells exhibited avoidance behavior (EC ). Each trial Cross-adaptation assays (Table 1)showthatall threeiso- represents 10 cells. A minimum of 6 trials was performed for forms of nociceptin cross-adapt with one another. However, each data point. nociceptin-adapted cells did not cross-adapt to PACAP-38, Pharmacological inhibition assays were performed simi- and PACAP-adapted cells did not cross-adapt to nociceptin. lar to the behavioral assays described previously. Aeft r being Since all three nociceptin isoforms cross-adapted to one washed in bueff r, cells were exposed to pharmacological another, implying a common signaling pathway, we used agents known to block specific signaling pathways and incu- 50𝜇 M nociceptin-NH2 in all subsequent pharmacological bated for 15 minutes to 2 hours. Cells were then transferred to and behavioral assays. a solution containing nociceptin at EC and then monitored Studies with pharmacological agents known to block for avoidance behavior. Each trial represents 10 cells. A G-protein signaling, tyrosine kinase signaling, and broad minimum of 6 trials was performed for each data point. spectrum kinase activity had no eeff ct on avoidance behavior International Journal of Peptides 3 Table 1: Nociceptin cross-adaptation studies. Cells were adapted to a given ligand by incubating them in that ligand for 10–15 minutes or until avoidance behavior ceased. Cells were then moved into another ligand and were scored positively or negatively for avoidance. Cross-adaptation with various analogues of nociceptin all show avoidance values that are at or below baseline (≤20%; [10]). However, cross- adaptation with the polycationic peptide, PACAP, does not cross-adapt with nociception, implying that nociception is using a pathway that is distinct from the previously described lysozyme/PACAP receptor [10, 15].𝑁 represents the number of trials conducted. Each trial consisted of 10 cells, which were individually scored as positive or negative for avoidance. Nociceptin Nociceptin-NH Nociceptin Arg Lys PACAP 1-38 2 14 15 9.2± 8.2 5± 8.3 0± 0 96.6± 5.8 Nociceptin 𝑁=13 𝑁=6 𝑁=6 𝑁=6 5± 7.5 16.9± 12.2 14.5± 12.4 100± 0 Nociceptin-NH 𝑁=9 𝑁=8 𝑁=12 𝑁=6 3.3± 5.8 13.3± 12.1 16.6± 16.3 91.25± 9.9 Nociceptin Arg Lys 14 15 𝑁=6 𝑁=6 𝑁=6 𝑁=8 97.5± 4.6 100±0100±013.3± 5.8 PACAP 1-38 𝑁=8 𝑁=10 𝑁=10 𝑁=6 100 100 80 80 60 60 40 40 0.1 1 10 100 1000 10000 0.1 1 10 100 1000 [Inhibitor] (𝜇 M) [Nociceptin] (𝜇 M) Figure 2: Calcium chelators inhibit the behavioral response to Figure 1: Nociceptin is a chemorepellent in Tetrahymena ther- 50𝜇 M nociceptin-NH2 in Tetrahymena thermophila. EGTA (closed mophila. Nociceptin (closed circles), nociceptin-NH2 (closed circles) reduces avoidance to 20% (near baseline) at a concentration squares), and nociceptin-Arg Lys (closed diamonds) all caused 14 15 of 50𝜇 M. The IC of EGTA is approximately 7.5𝜇 M. Thapsigargin avoidance in Tetrahymena thermophila.TheEC of each com- (open triangles) reduced avoidance by 50% at a concentration of pound was correlated with its charge. Nociceptin, which has a net 100𝜇 M; however, increasing the concentration to 300𝜇 M did not charge of +4, had an EC of 100𝜇 M. Nociceptin-NH2, which has cause a significant decrease in avoidance beyond that seen with a net charge of +5, had an EC of 50𝜇 M. Finally, nociceptin- 100𝜇 M thapsigargin.𝑁≥6 .𝑁 represents the number of trials Arg Lys , which had a net charge of +6, had an EC of 25𝜇 M. 14 15 100 conducted. Each trial consisted of 10 cells, which were individually 𝑁≥6 .𝑁 represents the number of trials conducted. Each trial scored as positive or negative for avoidance. consisted of 10 cells, which were individually scored as positive or negative for avoidance. in T. thermophila (Table 2). However, studies with the calcium (Figure 3). A nociceptin-NH2 concentration of just 5𝜇 M chelator, EGTA, and the SERCA ATPase inhibitor, thapsigar- was sufficient to elicit a depolarization of approximately gin, both aeff cted nociceptin avoidance ( Figure 2). A concen- 20 mV, though this concentration does not cause tration of 50𝜇 MEGTAwas sucffi ient to reduce avoidanceto behavioral avoidance above baseline levels in Tetrahymena a baseline avoidance of 20%. Thapsigargin, however, never (Figures 3(a) and 1). Fifty 𝜇 M nociceptin-NH2, which is the reduced avoidance to baseline under the conditions of our EC for behavioral avoidance in Tetrahymena,eliciteda assay. eTh highest concentration of thapsigargin we were able depolarization of approximately 40 mV (Figure 3(b)). The to achieve in our assay was 300𝜇 M. aTh psigargin reduced depolarization produced by 50𝜇 M nociceptin-NH2 was avoidance by 50% at a concentration of 100𝜇 M; however, eliminated by the addition of 1 mM EGTA to the external increasing the concentration to 300𝜇 Mdid notdecrease medium (Figure 3(c)). avoidance beyond that seen with 100𝜇 Mthapsigargin. J-113397, a competitive inhibitor of the human noci- Whole-cell electrophysiology studies indicate that ceptin receptor, inhibited the behavioral response to 50𝜇 M nociceptin-NH2 is a depolarizing signal in T. thermophila nociceptin-NH2 in Tetrahymena thermophila when applied Cells showing avoidance (%) Cells showing avoidance (%) 4 International Journal of Peptides 50 𝜇 M nociceptin-NH Buffer 5𝜇 M nociceptin-NH Buffer Buffer 2 50 𝜇 M +1 mM nociceptin-NH + EGTA 1 mM EGTA 1 min 1 min 1 min (a) (b) (c) Figure 3: Nociceptin-NH2 is a depolarizing signal in Tetrahymena thermophila.(a) 5𝜇 M nociceptin-NH2 causes a depolarization of approximately 20 mV, though this concentration does not often provoke a behavioral response in Tetrahymena. (b) 50𝜇 M nociceptin- NH2 causes a depolarization of approximately 40 mV. This concentration is the EC for behavioral avoidance in Tetrahymena. (c) eTh depolarization produced by 50𝜇 M nociceptin-NH2 is eliminated by the addition of 1 mM EGTA to the external medium, implying that calcium is involved in the depolarization. Table 2: Pharmacological inhibitors which act on G-protein mediated r eceptor pathways and tyrosine kinase pathways do not significantly impact nociception avoidance.𝑁 represents the number of trials conducted. Each trial consisted of 10 cells, which were individually scored as positive or negative for avoidance. Pharmacological inhibitor Pathway inhibited Percentage of cells avoiding nociceptin Control None 94.28± 5.34 50𝜇 M RpcAMPs Adenylyl cyclase 96.67± 5.16 1mM GDP-𝛽 -S G-proteins 100.0± 0.0 1𝜇 M U-73122 Phospholipase C 97.78± 4.40 1𝜇 M U-73345 Inactive analogue of U-73122 96.67± 5.16 10𝜇 M calphostin C Protein kinase C 96.0± 5.0 1 mM 1400 W NOS 96.0± 5.0 100𝜇 M tyrphostin 47 Receptor tyrosine kinases 92.5± 9.57 100𝜇 M AG126 Map kinase pathway 96.0± 6.99 150𝜇 M SU 6668 Receptor tyrosine kinases 91.66± 4.08 300𝜇 M apigenin Protein kinases 91.2± 6.4 3 mM H-9 Protein kinases 96.6± 5.1 extracellularly (Figure 4). Baseline avoidance to nociceptin compound was correlated with the charge, with the most was achieved by the addition of 50𝜇 M of J-113397. highly charged isoform having the lowest EC ,althoughall of the EC values were in a similar range. The correlation of lower EC values with a higher charge is consistent 4. Discussion 100 with what we have seen using other charged peptides in T. Ourresults conrfi med ourhypothesisthatall threenoci- thermophila. Forexample,whenwehaveusedvarious pep- tides derived from ACTH, the more highly charged peptides ceptin isoforms tested would serve as chemorepellents in T. thermophila (Figure 1). In addition, the EC of each caused avoidance at lower concentrations than did the less 10 mV 10 mV 10 mV International Journal of Peptides 5 100 associated pathways. None of these inhibitors blocked avoid- ance to nociceptin (Table 2), giving further evidence that the previously described polycation receptor is not being used in nociceptin signaling. This also differs from the vertebrate nociceptin receptor, which signals through G proteins [1]. 𝑖/𝑜 Since a tyrosine kinase has been implicated in GTP signaling in T. thermophila [18] as well as insulin signaling [19], we also tested a battery of protein kinase and tyrosine kinase inhibitors to determine whether nociceptin signaling wouldbeinhibited.Noneofthese inhibitors aeff cted noci- ceptin signaling (Table 2). Interestingly, genomic studies of Tetrahymena [20]shownoevidence of thepresenceofa tyrosine kinase in this organism. 0.1 1 10 100 Since a calcium-based depolarization is elicited by the [J113397] (𝜇 M) addition of lysozyme [12]aswellasthe lysozyme frag- ment, CB [8], to T. thermophila, we wished to determine Figure 4: J-113397, a competitive inhibitor of the human nociceptin whether calcium was involved in nociceptin signaling in this receptor, inhibits the behavioral response to 50𝜇 M nociceptin-NH2 organism. Studies with the external calcium chelator, EGTA in Tetrahymena thermophila when applied extracellularly. The IC (Figure 2) indicated that extracellular calcium was necessary of this compound is approximately 5𝜇 M.𝑁≥6 .𝑁 represents the for behavioral avoidance to nociceptin, since concentrations number of trials conducted. Each trial consisted of 10 cells, which were individually scored as positive or negative for avoidance. of EGTA above 50𝜇 M reduced avoidance down to baseline. Baseline avoidance in this organism is determined by count- ing the number of cells that show avoidance behavior when transferred from one well of buffer to another well of the same bueff r [ 10]. The SERCA ATPase inhibitor, thapsigargin, highly charged peptides [11]. In addition, our previous studies with PACAP and VIP [15]showthatPACAP is eeff ctive at was used to determine whether internal calcium stores were causing avoidance at a 1000-fold lower concentration than required in order for avoidance to occur. As seen in Figure 2, exposure of cells to 100𝜇 M thapsigargin reduced avoidance VIP, though presumably acting through the same receptor and/or signaling pathway. eTh isoform of PACAP that we by approximately 50%. However, the avoidance response was used in the 2003 study, PACAP-38-NH2, has a net charge not completely inhibited, indicating that while intracellular of +11 at pH 7.0, while VIP has a net charge of just +4 at calcium may play a role in avoidance, lack of intracellular thesamepH. Whilefactors otherthanchargeare certainly calcium stores depleted by thapsigargin may be partially involved in the interaction between these peptides and their compensated for by allowing extracellular calcium into the putative receptor, it is highly probable that charge is playing cytosol. Notably, the thapsigargin concentration used in this a role in these interactions, possibly by increasing the ani ffi ty study was much higher than what we used in a previous study [18], in which only 1 nM thapsigargin was necessary in of ligand for its receptor. In the case of nociceptin, the charge differences were relatively small as were the differences in order to block the behavioral response to GTP. This is further EC . evidence that extracellular calcium is primarily responsible for nociceptin avoidance. Calcium is not necessary for avoid- Cells acclimated to nociception within 10–15 minutes of rfi st being exposed to it (not shown). All isoforms of ance to all peptides, however, since avoidance of netrin-1, nociceptin were cross-adapted to one another, indicating that semaphorin 3C, and fragments of ACTH is unaeff cted by all forms of nociceptin were using the same receptor and/or addition of either EGTA or thapsigargin [11]. signaling pathway. This is similar to what has previously Whole-cell electrophysiology studies indicate that noci- been shown for lysozyme [8] and PACAP/VIP [15]. Since ceptin causes a depolarization in T. thermophila (Figure 3), PACAP, lysozyme, and VIP appear to share a common even at concentrations that normally do not cause a behav- receptor [10, 15], we cross-adapted cells to nociceptin and ioral response in this organism (Figures 3(a) and 1). When the EC of nociceptin-NH2 was used, the amplitude of PACAP to determine whether nociceptin was using the same receptor/signaling pathway as the three previously studied the depolarization increased (Figure 3(b)). Finally, we were polycationic ligands. As Table 1 shows, PACAP-adapted cells able to remove the depolarization by the addition of EGTA to the external medium (Figure 3(c)), implying that calcium did not cross-adapt to nociceptin and nociceptin-adapted cells did not cross-adapt to PACAP. This indicates that is involved in the depolarization. This is similar to the nociceptin signals through a pathway that does not involve previously described responses to lysozyme [12]and the the previously described polycation receptor. lysozyme fragment, CB2 [9]. The previously studied PACAP response appears to be The involvement of calcium in nociceptin avoidance in T. thermophila is rather different from the human response mediated through a G-protein-coupled receptor which uses adenylyl cyclase, phospholipase C, and nitric oxide synthase to nociceptin, which involves closing calcium channels [10, 13–15]. In order to further ascertain whether nociceptin [5]. However, we did use J-113397, which is a competitive inhibitor of the human nociceptin receptor [21], in order to was using a separate signaling pathway, we used pharma- cological inhibitors to block G-protein-linked receptors and determine if it could also block T. thermophila avoidance Cells showing avoidance (%) 6 International Journal of Peptides to nociceptin. As shown in Figure 4,50𝜇 M J-113397 was References eeff ctive in reducing avoidance to baseline. This drug had no [1] B. E. Hawes, M. P. Graziano, and D. G. Lambert, “Cellular effect on avoidance to ACTH fragments (data not shown), actions of nociceptin: transduction mechanisms,” Peptides,vol. suggesting that the response was specific to nociceptin. While 21,no. 7, pp.961–967,2000. we have not identified the receptor or signaling pathway that [2] E. Hashiba, C. Harrison, G. 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Nociceptin Signaling Involves a Calcium-Based Depolarization in Tetrahymena thermophila

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Copyright © 2013 Thomas Lampert et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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1687-9767
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10.1155/2013/573716
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Hindawi Publishing Corporation International Journal of Peptides Volume 2013, Article ID 573716, 7 pages http://dx.doi.org/10.1155/2013/573716 Research Article Nociceptin Signaling Involves a Calcium-Based Depolarization in Tetrahymena thermophila 1 2 2 2 Thomas Lampert, Cheryl Nugent, John Weston, Nathanael Braun, and Heather Kuruvilla Department of Biological Sciences, State University of New York at Buaff lo, 109 Cooke Hall, Buaff lo,NY14260,USA Department of Science and Mathematics, Cedarville University, 251 North Main Street, Cedarville, OH 45314, USA Correspondence should be addressed to Heather Kuruvilla; heatherkuruvilla@cedarville.edu Received 7 March 2013; Accepted 9 April 2013 Academic Editor: Hubert Vaudry Copyright © 2013 Thomas Lampert et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tetrahymena thermophila are free-living, ciliated eukaryotes. eTh ir 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 𝜇 M nociceptin-NH2 causes a sustained depolarization of approximately 40 mV, which is eliminated by the addition of extracellular EGTA. 1. Introduction their environment. This allows them to find food and possibly to escape predation [6]. A recent review by Csaba [7] details Nociceptin/orphanin FQ (hereaer ft referred to as nociceptin) the response of T. thermophila to a number of chemoat- is a peptide involved in vertebrate pain signaling. The tractants and chemorepellents, including their response to endogenous receptor for this ligand is ORL-1/NCR [1, 2]. many vertebrate hormones. Indeed, T. thermophila appear A number of signaling pathways have been implicated in to synthesize and respond to a number of vertebrate hor- vertebrate nociceptin signaling. A partial listing of molecules mones, including serotonin, melatonin, adrenocorticotropic involved in this signaling cascade would include G proteins 𝑖/𝑜 hormone, and insulin [7]. [1], neuronal nitric oxide synthase (nNOS) [3], and Erk- A number of chemorepellents which have been charac- dependent signaling [4]. In addition, signaling through the terized in T. thermophila are polycationic peptides, including nociceptin receptor induces a reduction in calcium influx via lysozyme [8], thelysozymefragmentCB2 [9], PACAP [10], P/Q-type calciumchannelsinrat brain[5]. and nociceptive peptides including bradykinin and substance Tetrahymena thermophila are free-living, unicellular P[11]. Lysozyme signaling involves a calcium-based depolar- eukaryotes. While T. thermophila do not feel pain, they are ization [12]. Lysozyme and PACAP appear to share a signaling capable of sensing chemoattractants and chemorepellents in pathway [9], which involves cAMP and phospholipase C 2 International Journal of Peptides [13], as well as NOS and cGMP [14]. A related peptide, Cross-adaptation assays were performed as previously VIP, also uses these signaling pathways and cross-adapts described [10, 17]. Briefly, 300 𝜇 Lofcells were placed into with lysozyme and PACAP, suggesting that Tetrahymena are the rfi st well of a 3-well microtiter plate. Cells were then signaling through a generalized polycation receptor [15]. individually transferred to the second well of the 3-well Nociceptin is a polycationic peptide that is commercially microtiter plate, which contained a repellent. eTh cells were available in three different isoforms. Nociceptin carries a allowed to adapt to this repellent for 10–15 minutes or until charge of +4 at pH 7.0, while nociceptin-NH2 carries a charge cells showed baseline avoidance (an avoidance of no more of +5 at pH 7.0. Nociceptin-Arg Lys carries a charge of +6 than 20%). Cells were then individually transferred to the 14 15 at pH 7.0. Our hypothesis was that all three of the nociceptin third well of the 3-well microtiter plate, which contained the analogues would be chemorepellents in T. thermophila and repellent to be tested for cross-adaptation and monitored for that more highly charged nociceptin isoforms will have a avoidance behavior. Data which showed 20% or fewer cells lower EC in behavioral assays than isoforms which carry exhibiting avoidance was considered “baseline avoidance.” alessercharge. Baseline avoidance is the number of cells in our assay which show avoidance behavior when being transferred from one well containing buffer to another well containing the same 2. Materials and Methods buffer and usually ranges from 5 to 20%. Cells exhibiting baseline avoidance in response to this assay were considered to be cross-adapted. Each trial represents 10 cells. A minimum 2.1. Cell Cultures. Tetrahymena thermophila,strainB2086,a of 6 trials was performed for each data point. generous gift from Hennessey [ 6] (SUNY Buffalo), was used for all of the experiments. Cells were grown at 25 Cinthe axenic medium of Dentler [16], without shaking or addition 2.4. Electrophysiology. Standard one-electrode whole-cell of antibiotics. Two-day old cell cultures were used for all membrane potential recordings were recorded as the pre- behavioral assays described below. viously reported procedures in Tetrahymena thermophila [9, 12]. eTh recording bueff r contained were carried out in a buffer of pH 7.0 containing 10 mM Trizma base, 2.2. Chemicals and Solutions. Behavioral assays were car- 0.5 mM MOPS, and 1 mM CaCl . Membrane potentials ried out in a buffer of pH 7.0 containing 10 mM Trizma were displayed on a digital oscilloscope and retained on base,0.5mM MOPS,and 50𝜇 M CaCl . All repellents and a chart recorder during continuous bath perfusion at a inhibitors used were dissolved in this bueff r. rate of approximately 20.0 mL/min. The recording bath had All nociceptin isoforms, thapsigargin, J-113397, and a volume of approximately 1 mL. Solutions were changed EGTA, were purchased from Tocris Biosciences, Bristol, UK. by switching valves connected either to bueff r or to the experimental solution without changing the flow rate of the 2.3. Behavioral Assays. Behavioral assays were carried out perfusion system. as previously described [8, 10, 17]. Ten milliliters of T. thermophila culture was washed by centrifugation in a clinical centrifuge at high speed, and the pellet was reconstituted in 10 mL bueff r. This wash step was repeated twice, and cells were 3. Results reconstituted in 5 mL of buffer for use in behavioral assays. To perform the behavioral assays, 300𝜇 Lofcellsuspensionwas All isoforms of nociceptin were chemorepellents in T. ther- transferred to the rfi st well of a microtiter plate. Cells were mophila (Figure 1). Nociceptin, which has a charge of +4 at then transferred individually using a micropipette into the our assay pH of 7.0, had an EC of 100𝜇 Minour behavioral second well of the microtiter plate, which contained 300𝜇 L assay. Nociceptin-NH2, which has a charge of +5 under assay of buffer as a control. Cells were then transferred to a third conditions, had an EC of 50𝜇 M, while nociceptin-Arg - 100 14 well containing 300𝜇 L of nociceptin. Behavior of the cells was Lys which has a charge of +6 under assay conditions had observed for the rfi st 5 seconds aeft r transfer to the third well, an EC of 25𝜇 M. Avoidance was observed for 1–5 seconds and the percentage of cells exhibiting avoidance behavior was but was seen for as long as 10–15 minutes (not shown). Aeft r noted. Varying concentrations of each peptide were used until cells acclimated to the nociceptin, they returned to forward we determined the minimum concentration at which 90% swimming. of the cells exhibited avoidance behavior (EC ). Each trial Cross-adaptation assays (Table 1)showthatall threeiso- represents 10 cells. A minimum of 6 trials was performed for forms of nociceptin cross-adapt with one another. However, each data point. nociceptin-adapted cells did not cross-adapt to PACAP-38, Pharmacological inhibition assays were performed simi- and PACAP-adapted cells did not cross-adapt to nociceptin. lar to the behavioral assays described previously. Aeft r being Since all three nociceptin isoforms cross-adapted to one washed in bueff r, cells were exposed to pharmacological another, implying a common signaling pathway, we used agents known to block specific signaling pathways and incu- 50𝜇 M nociceptin-NH2 in all subsequent pharmacological bated for 15 minutes to 2 hours. Cells were then transferred to and behavioral assays. a solution containing nociceptin at EC and then monitored Studies with pharmacological agents known to block for avoidance behavior. Each trial represents 10 cells. A G-protein signaling, tyrosine kinase signaling, and broad minimum of 6 trials was performed for each data point. spectrum kinase activity had no eeff ct on avoidance behavior International Journal of Peptides 3 Table 1: Nociceptin cross-adaptation studies. Cells were adapted to a given ligand by incubating them in that ligand for 10–15 minutes or until avoidance behavior ceased. Cells were then moved into another ligand and were scored positively or negatively for avoidance. Cross-adaptation with various analogues of nociceptin all show avoidance values that are at or below baseline (≤20%; [10]). However, cross- adaptation with the polycationic peptide, PACAP, does not cross-adapt with nociception, implying that nociception is using a pathway that is distinct from the previously described lysozyme/PACAP receptor [10, 15].𝑁 represents the number of trials conducted. Each trial consisted of 10 cells, which were individually scored as positive or negative for avoidance. Nociceptin Nociceptin-NH Nociceptin Arg Lys PACAP 1-38 2 14 15 9.2± 8.2 5± 8.3 0± 0 96.6± 5.8 Nociceptin 𝑁=13 𝑁=6 𝑁=6 𝑁=6 5± 7.5 16.9± 12.2 14.5± 12.4 100± 0 Nociceptin-NH 𝑁=9 𝑁=8 𝑁=12 𝑁=6 3.3± 5.8 13.3± 12.1 16.6± 16.3 91.25± 9.9 Nociceptin Arg Lys 14 15 𝑁=6 𝑁=6 𝑁=6 𝑁=8 97.5± 4.6 100±0100±013.3± 5.8 PACAP 1-38 𝑁=8 𝑁=10 𝑁=10 𝑁=6 100 100 80 80 60 60 40 40 0.1 1 10 100 1000 10000 0.1 1 10 100 1000 [Inhibitor] (𝜇 M) [Nociceptin] (𝜇 M) Figure 2: Calcium chelators inhibit the behavioral response to Figure 1: Nociceptin is a chemorepellent in Tetrahymena ther- 50𝜇 M nociceptin-NH2 in Tetrahymena thermophila. EGTA (closed mophila. Nociceptin (closed circles), nociceptin-NH2 (closed circles) reduces avoidance to 20% (near baseline) at a concentration squares), and nociceptin-Arg Lys (closed diamonds) all caused 14 15 of 50𝜇 M. The IC of EGTA is approximately 7.5𝜇 M. Thapsigargin avoidance in Tetrahymena thermophila.TheEC of each com- (open triangles) reduced avoidance by 50% at a concentration of pound was correlated with its charge. Nociceptin, which has a net 100𝜇 M; however, increasing the concentration to 300𝜇 M did not charge of +4, had an EC of 100𝜇 M. Nociceptin-NH2, which has cause a significant decrease in avoidance beyond that seen with a net charge of +5, had an EC of 50𝜇 M. Finally, nociceptin- 100𝜇 M thapsigargin.𝑁≥6 .𝑁 represents the number of trials Arg Lys , which had a net charge of +6, had an EC of 25𝜇 M. 14 15 100 conducted. Each trial consisted of 10 cells, which were individually 𝑁≥6 .𝑁 represents the number of trials conducted. Each trial scored as positive or negative for avoidance. consisted of 10 cells, which were individually scored as positive or negative for avoidance. in T. thermophila (Table 2). However, studies with the calcium (Figure 3). A nociceptin-NH2 concentration of just 5𝜇 M chelator, EGTA, and the SERCA ATPase inhibitor, thapsigar- was sufficient to elicit a depolarization of approximately gin, both aeff cted nociceptin avoidance ( Figure 2). A concen- 20 mV, though this concentration does not cause tration of 50𝜇 MEGTAwas sucffi ient to reduce avoidanceto behavioral avoidance above baseline levels in Tetrahymena a baseline avoidance of 20%. Thapsigargin, however, never (Figures 3(a) and 1). Fifty 𝜇 M nociceptin-NH2, which is the reduced avoidance to baseline under the conditions of our EC for behavioral avoidance in Tetrahymena,eliciteda assay. eTh highest concentration of thapsigargin we were able depolarization of approximately 40 mV (Figure 3(b)). The to achieve in our assay was 300𝜇 M. aTh psigargin reduced depolarization produced by 50𝜇 M nociceptin-NH2 was avoidance by 50% at a concentration of 100𝜇 M; however, eliminated by the addition of 1 mM EGTA to the external increasing the concentration to 300𝜇 Mdid notdecrease medium (Figure 3(c)). avoidance beyond that seen with 100𝜇 Mthapsigargin. J-113397, a competitive inhibitor of the human noci- Whole-cell electrophysiology studies indicate that ceptin receptor, inhibited the behavioral response to 50𝜇 M nociceptin-NH2 is a depolarizing signal in T. thermophila nociceptin-NH2 in Tetrahymena thermophila when applied Cells showing avoidance (%) Cells showing avoidance (%) 4 International Journal of Peptides 50 𝜇 M nociceptin-NH Buffer 5𝜇 M nociceptin-NH Buffer Buffer 2 50 𝜇 M +1 mM nociceptin-NH + EGTA 1 mM EGTA 1 min 1 min 1 min (a) (b) (c) Figure 3: Nociceptin-NH2 is a depolarizing signal in Tetrahymena thermophila.(a) 5𝜇 M nociceptin-NH2 causes a depolarization of approximately 20 mV, though this concentration does not often provoke a behavioral response in Tetrahymena. (b) 50𝜇 M nociceptin- NH2 causes a depolarization of approximately 40 mV. This concentration is the EC for behavioral avoidance in Tetrahymena. (c) eTh depolarization produced by 50𝜇 M nociceptin-NH2 is eliminated by the addition of 1 mM EGTA to the external medium, implying that calcium is involved in the depolarization. Table 2: Pharmacological inhibitors which act on G-protein mediated r eceptor pathways and tyrosine kinase pathways do not significantly impact nociception avoidance.𝑁 represents the number of trials conducted. Each trial consisted of 10 cells, which were individually scored as positive or negative for avoidance. Pharmacological inhibitor Pathway inhibited Percentage of cells avoiding nociceptin Control None 94.28± 5.34 50𝜇 M RpcAMPs Adenylyl cyclase 96.67± 5.16 1mM GDP-𝛽 -S G-proteins 100.0± 0.0 1𝜇 M U-73122 Phospholipase C 97.78± 4.40 1𝜇 M U-73345 Inactive analogue of U-73122 96.67± 5.16 10𝜇 M calphostin C Protein kinase C 96.0± 5.0 1 mM 1400 W NOS 96.0± 5.0 100𝜇 M tyrphostin 47 Receptor tyrosine kinases 92.5± 9.57 100𝜇 M AG126 Map kinase pathway 96.0± 6.99 150𝜇 M SU 6668 Receptor tyrosine kinases 91.66± 4.08 300𝜇 M apigenin Protein kinases 91.2± 6.4 3 mM H-9 Protein kinases 96.6± 5.1 extracellularly (Figure 4). Baseline avoidance to nociceptin compound was correlated with the charge, with the most was achieved by the addition of 50𝜇 M of J-113397. highly charged isoform having the lowest EC ,althoughall of the EC values were in a similar range. The correlation of lower EC values with a higher charge is consistent 4. Discussion 100 with what we have seen using other charged peptides in T. Ourresults conrfi med ourhypothesisthatall threenoci- thermophila. Forexample,whenwehaveusedvarious pep- tides derived from ACTH, the more highly charged peptides ceptin isoforms tested would serve as chemorepellents in T. thermophila (Figure 1). In addition, the EC of each caused avoidance at lower concentrations than did the less 10 mV 10 mV 10 mV International Journal of Peptides 5 100 associated pathways. None of these inhibitors blocked avoid- ance to nociceptin (Table 2), giving further evidence that the previously described polycation receptor is not being used in nociceptin signaling. This also differs from the vertebrate nociceptin receptor, which signals through G proteins [1]. 𝑖/𝑜 Since a tyrosine kinase has been implicated in GTP signaling in T. thermophila [18] as well as insulin signaling [19], we also tested a battery of protein kinase and tyrosine kinase inhibitors to determine whether nociceptin signaling wouldbeinhibited.Noneofthese inhibitors aeff cted noci- ceptin signaling (Table 2). Interestingly, genomic studies of Tetrahymena [20]shownoevidence of thepresenceofa tyrosine kinase in this organism. 0.1 1 10 100 Since a calcium-based depolarization is elicited by the [J113397] (𝜇 M) addition of lysozyme [12]aswellasthe lysozyme frag- ment, CB [8], to T. thermophila, we wished to determine Figure 4: J-113397, a competitive inhibitor of the human nociceptin whether calcium was involved in nociceptin signaling in this receptor, inhibits the behavioral response to 50𝜇 M nociceptin-NH2 organism. Studies with the external calcium chelator, EGTA in Tetrahymena thermophila when applied extracellularly. The IC (Figure 2) indicated that extracellular calcium was necessary of this compound is approximately 5𝜇 M.𝑁≥6 .𝑁 represents the for behavioral avoidance to nociceptin, since concentrations number of trials conducted. Each trial consisted of 10 cells, which were individually scored as positive or negative for avoidance. of EGTA above 50𝜇 M reduced avoidance down to baseline. Baseline avoidance in this organism is determined by count- ing the number of cells that show avoidance behavior when transferred from one well of buffer to another well of the same bueff r [ 10]. The SERCA ATPase inhibitor, thapsigargin, highly charged peptides [11]. In addition, our previous studies with PACAP and VIP [15]showthatPACAP is eeff ctive at was used to determine whether internal calcium stores were causing avoidance at a 1000-fold lower concentration than required in order for avoidance to occur. As seen in Figure 2, exposure of cells to 100𝜇 M thapsigargin reduced avoidance VIP, though presumably acting through the same receptor and/or signaling pathway. eTh isoform of PACAP that we by approximately 50%. However, the avoidance response was used in the 2003 study, PACAP-38-NH2, has a net charge not completely inhibited, indicating that while intracellular of +11 at pH 7.0, while VIP has a net charge of just +4 at calcium may play a role in avoidance, lack of intracellular thesamepH. Whilefactors otherthanchargeare certainly calcium stores depleted by thapsigargin may be partially involved in the interaction between these peptides and their compensated for by allowing extracellular calcium into the putative receptor, it is highly probable that charge is playing cytosol. Notably, the thapsigargin concentration used in this a role in these interactions, possibly by increasing the ani ffi ty study was much higher than what we used in a previous study [18], in which only 1 nM thapsigargin was necessary in of ligand for its receptor. In the case of nociceptin, the charge differences were relatively small as were the differences in order to block the behavioral response to GTP. This is further EC . evidence that extracellular calcium is primarily responsible for nociceptin avoidance. Calcium is not necessary for avoid- Cells acclimated to nociception within 10–15 minutes of rfi st being exposed to it (not shown). All isoforms of ance to all peptides, however, since avoidance of netrin-1, nociceptin were cross-adapted to one another, indicating that semaphorin 3C, and fragments of ACTH is unaeff cted by all forms of nociceptin were using the same receptor and/or addition of either EGTA or thapsigargin [11]. signaling pathway. This is similar to what has previously Whole-cell electrophysiology studies indicate that noci- been shown for lysozyme [8] and PACAP/VIP [15]. Since ceptin causes a depolarization in T. thermophila (Figure 3), PACAP, lysozyme, and VIP appear to share a common even at concentrations that normally do not cause a behav- receptor [10, 15], we cross-adapted cells to nociceptin and ioral response in this organism (Figures 3(a) and 1). When the EC of nociceptin-NH2 was used, the amplitude of PACAP to determine whether nociceptin was using the same receptor/signaling pathway as the three previously studied the depolarization increased (Figure 3(b)). Finally, we were polycationic ligands. As Table 1 shows, PACAP-adapted cells able to remove the depolarization by the addition of EGTA to the external medium (Figure 3(c)), implying that calcium did not cross-adapt to nociceptin and nociceptin-adapted cells did not cross-adapt to PACAP. This indicates that is involved in the depolarization. This is similar to the nociceptin signals through a pathway that does not involve previously described responses to lysozyme [12]and the the previously described polycation receptor. lysozyme fragment, CB2 [9]. The previously studied PACAP response appears to be The involvement of calcium in nociceptin avoidance in T. thermophila is rather different from the human response mediated through a G-protein-coupled receptor which uses adenylyl cyclase, phospholipase C, and nitric oxide synthase to nociceptin, which involves closing calcium channels [10, 13–15]. In order to further ascertain whether nociceptin [5]. However, we did use J-113397, which is a competitive inhibitor of the human nociceptin receptor [21], in order to was using a separate signaling pathway, we used pharma- cological inhibitors to block G-protein-linked receptors and determine if it could also block T. thermophila avoidance Cells showing avoidance (%) 6 International Journal of Peptides to nociceptin. As shown in Figure 4,50𝜇 M J-113397 was References eeff ctive in reducing avoidance to baseline. This drug had no [1] B. E. Hawes, M. P. Graziano, and D. G. Lambert, “Cellular effect on avoidance to ACTH fragments (data not shown), actions of nociceptin: transduction mechanisms,” Peptides,vol. suggesting that the response was specific to nociceptin. While 21,no. 7, pp.961–967,2000. we have not identified the receptor or signaling pathway that [2] E. Hashiba, C. Harrison, G. Calo’ et al., “Characterisation nociceptin is using in T. thermophila,these data suggestthat and comparison of novel ligands for the nociceptin/orphanin there may be commonalities between the human nociceptin FQ receptor,” Naunyn-Schmiedeberg’s Archives of Pharmacology, receptor and a possible nociceptin-binding protein in T. vol. 363, no. 1, pp. 28–33, 2001. thermophila. [3] L. Xu, E. Okuda-Ashitaka, S. Matsumura et al., “Signal pathways In summary, we have shown that nociceptin is a chemore- coupled to activation of neuronal nitric oxide synthase in the pellent in Tetrahymena which elicits a depolarization. It spinal cord by nociceptin/orphanin FQ,” Neuropharmacology, does not act through the previously described polycation vol. 52,no. 5, pp.1318–1325,2007. receptor nor does it signal through a G-protein-mediated [4] C. Goeldner, D. Reiss, J. Wichmann, H. Meziane, B. L. Kieffer, receptor like the vertebrate nociceptin receptor. However, and A. M. Ouagazzal, “Nociceptin receptor impairs recogni- the J113397 studies imply that Tetrahymena may possess tion memory via interaction with NMDA receptor-dependent some type of receptor that shares binding characteristics mitogen-activated protein kinase/extracellular signal-regulated with the human nociceptin receptor. Further studies may kinase signaling in the hippocampus,” Journal of Neuroscience, help elucidate the signaling mechanisms used in nociceptin vol. 28, no. 9, pp. 2190–2198, 2008. avoidance in T. thermophila. If the receptor is identified, [5] H.S.Gompf,M.G.Moldavan, R. P. Irwin, andC.N.Allen, comparisons between the human nociceptin receptor and the “Nociceptin/orphanin FQ (N/OFQ) inhibits excitatory and unknown nociceptin-sensing mechanism in T. thermophila inhibitory synaptic signaling in the suprachiasmatic nucleus (SCN),” Neuroscience,vol.132,no. 4, pp.955–965,2005. would be instructive. [6] T. M. 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