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Antagonistic effects of [Nphe1]nociceptin(1–13)NH2 on nociceptin receptor mediated inhibition of cAMP formation in Chinese ovary hamster cells stably expressing the recombinant human nociceptin receptor
Neuroscience Letters 278 (2000) 109±112 www.elsevier.com/locate/neulet
Antagonistic effects of [Nphe 1]nociceptin(1±13)NH2 on nociceptin receptor mediated inhibition of cAMP formation in Chinese hamster ovary cells stably expressing the recombinant human nociceptin receptor Y. Hashimoto a, G. Calo' b, R. Guerrini c, G. Smith a, D.G. Lambert a,* a
University Department of Anaesthesia and Pain Management, Leicester Royal In®rmary, Leicester, LE1 5WW, UK Department of Experimental and Clinical Medicine, Section of Pharmacology, University of Ferrara, via Fossato di Mortara, 17, 44100 Ferrara, Italy c Department of Pharmaceutical Sciences, University of Ferrara, via Fossato di Mortara, 17, 44100 Ferrara, Italy
b
Received 20 September 1999; received in revised form 15 November 1999; accepted 15 November 1999
Abstract Nociceptin/orphanin FQ (NC) is the endogenous ligand for the nociceptin receptor (NCR) which is negatively coupled to adenylyl cyclase to inhibit the formation of cAMP. In this study we describe the inhibitory action of the novel NC analogue, [Nphe 1]nociceptin(1±13)NH2 on cAMP formation in Chinese hamster ovary cells expressing the human NCR. NC, NC(1±13)NH2, the pseudopeptides [Phe 1c(CH2±NH)Gly 2]NC(1±17)NH2 and [Phe 1c(CH2±NH)Gly 2]NC(1±13)NH2, the hexapeptide, acetyl-Arg-Tyr-Tyr-Arg-Trp-Lys-NH2 and buprenorphine all produced a concentration dependent inhibition of forskolin stimulated cAMP formation. This inhibition was competitively reversed by [Nphe 1]NC(1±13)NH2 with essentially identical pA2 values (6.12±6.48). [Nphe 1]NC(1±13)NH2 showed per se a negligible residual agonist activity (a , 0.15). q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nociceptin/orphanin FQ; Antagonist; [Nphe1]nociceptin(1±13)NH2; cAMP
Nociceptin/orphanin FQ (NC) is the endogenous ligand for the nociceptin receptor (NCR) [17,27]. NCR is coupled via the inhibitory guanine nucleotide binding protein (Gprotein) Gi/o to adenylyl cyclase [17,27], potassium channels [11,24], and calcium channels [12,15]. NCR activation leads to reduced cAMP formation, an enhanced outward K 1 conductance and closing of voltage sensitive Ca 21 channels. Whilst there is reasonable structural homology between opioid and NCR and similarity in the transduction pathways employed the physiological roles of these systems differ. Opioids produce classic antinociceptive responses. NC produces hyperalgesia or anti-opioid actions following intracerebroventricular injection and antinociception when administered intrathecally [17,27]. Thus, the precise role of NC in nociception remains to be resolved. There is currently a paucity of compounds active at the NCR and the lack of a selective, competitive antagonist has * Corresponding author. Tel.: 144-116-258-5291; fax: 144-116285-4487. E-mail address: [email protected] (D.G. Lambert)
clearly hampered the characterization of the NC/NCR system. Ligands active at NCR include naloxone benzoylhydrazone [18], rimcazole and carbamapentane [16] and Mr2266 [1]. All are relatively weak and do not display selectivity for NCR. We have recently reported the activity of the pseudopeptide [Phe 1c(CH2±NH)Gly 2]Nociceptin(1±13)NH2 ([F/ G]NC(1±13)NH2) in the guinea pig ileum and mouse vas deferens where an antagonist action at the NCR was observed [14]. However, we and others have also shown that [F/G]NC(1±13)NH2 inhibited forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells (CHO) stably expressing the nociceptin receptor [5,20,25] and inhibited K 1 evoked-glutamate release from rat cerebrocortical slices [20] indicating an agonist action. [F/ G]NC(1±13)NH2 is clearly of limited value in the elucidation of the role of NC/NC receptor system in the central nervous system. Earlier this year we reported to the International Narcotics Research Conference meeting the characteristics of a novel NC analogue, [Nphe 1]nociceptin(1±13)NH2
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Y. Hashimoto et al. / Neuroscience Letters 278 (2000) 109±112
([Nphe 1]NC(1±13)NH2) in a variety of preparations and have noted a (weak) competitive antagonist pro®le, essentially devoid of any agonist activity and with good selectivity for NCR over classical m, d and k opioid receptors [7]. In this study we have further characterized the antagonistic activity of [Nphe 1]NC(1±13)NH2 against a battery of NCR ligands; NC, NC(1±13)NH2, [F/G]NC(1±17)NH2, [F/ G]NC(1±13)NH2 [6,14,20,23], CAM6369 (acetyl-Arg-TyrTyr-Arg-Trp-Lys-NH2 [13,19]), and buprenorphine [25]. The chemicals and their sources were as follows; NC, NC(1±13)NH2, [F/G]NC(1±17)NH2, [F/G]NC(1±13)NH2 and [Nphe 1]NC(1±13)NH2 were synthesized at one of our institutes as previously described [6,23]. CAM6369 was a gift from Drs. D. Smart and A.T. McKnight (Parke±Davis Neuroscience Research Centre, Cambridge, UK). Cell culture medium (Dulbecco's Modi®ed Eagles medium (DMEM) and Hams F12), foetal calf serum, hygromycin B, and G418 were from Gibco (Paisley, UK). [2,8± 3H]cAMP (28.4 Ci/mmol) was purchased from NEN DuPont (Boston, MA, USA). HEPES was from USB (Cleveland, OH, USA). All other reagents were from Sigma Chemical Co. (Poole, UK). CHO cells stably expressing the human nociceptin receptor, CHONCR (transfected using pCIN5), were obtained from Dr. F. Marshall and Mrs. N. Bevan of Glaxo±Wellcome, Stevenage, Hearts, UK. CHONCR cells expressing 1.8 pmol/mg protein NCR [20] were cultured in DMEM/Hams F12 medium (v/v 50:50) supplemented with foetal bovine serum 5% (v/v), G418 200 mg/ml, and hygromycin B 200 mg/ml at 378C in 5% CO2/humidi®ed air. Cultures were passaged twice weekly and used for investigation when con¯uent (3±4 days). Cells were harvested with 10 mM HEPES buffered saline/0.05% EDTA, pH 7.4, washed twice with and resuspended in Krebs/HEPES buffer of the following composition; (mM): Na 1 (143.3), K 1 (4.7), Ca 21 (2.15), Mg 21 (1.2), Cl 2 (125.6), H2PO422 (1.2), SO422 (1.2), Glucose (11.7), and HEPES (10), BSA 0.5%, pH 7.4 with 10 M NaOH. cAMP formation was measured in 0.3 ml volumes of whole cell suspensions in the presence of isobutylmethylxanthine (IBMX; 1 mM) and forskolin (FSK: 1 mM). Peptidase inhibitors were not used as these did not in¯uence the response to NC (data not shown). NCR agonists were included in various concentrations in order to obtain a full concentration response curve. In some experiments 10mM [Nphe 1]NC(1±13)NH2 was included. After 15 min of incubation at 378C, reactions were terminated and cAMP extracted by the addition of HCl (10 M). The reaction mixture was neutralized with NaOH (10 M) and Tris±HCl (1 M, pH 7.4). cAMP mass was measured in the supernatants using a protein binding assay as described by Okawa et al. [20]. All data are expressed as mean ^ SEM. The concentration of each agonist producing half maximal inhibition of cAMP formation (EC50) was obtained by computer-assisted curve ®tting of individual curves. All curve ®tting was performed using PRISM-V2.0 (GraphPad software, San
Diego, CA, USA). Antagonist af®nities are expressed in terms of pA2, which is the negative logarithm to base 10 of the antagonist molar concentration that makes it necessary to double the agonist concentration to elicit the original submaximal response. pA2 was calculated using the following formula: pA2 2log10 CR-1=antagonist, where CR is the ratio of EC50 in the presence and absence of antagonist. A slope of unity of the Schild regression line against NC has been reported previously [7] and is therefore assumed for the additional agonists examined here. All agents examined inhibited forskolin-stimulated cAMP accumulation in CHONCR cells in a concentrationdependent fashion with maximal inhibition of around 100% (Fig. 1A±F). [Nphe 1]NC(1±13)NH2 produced a competitive antagonism of each agonist response resulting in a parallel rightward shift of the concentration response curve. Maximal inhibition and pEC50 values in the absence and presence of [Nphe 1]NC(1±13)NH2 along with the estimated pA2 values are presented in Table 1. There was a small direct inhibition of cAMP formation in response to [Nphe 1]NC(1±13)NH2 alone (14.4 ^ 0.8%) which did not differ from our previous report [7]. We report that [Nphe 1]NC(1±13)NH2 acts as a competitive antagonist against a range of NCR agonists with essentially identical pA2 values. The pA2 against NC inhibition of cAMP formation is in agreement with the value reported by us previously [7]. Moreover, the effects of NC(1±13)NH2 (the template used for both [Nphe 1]NC(1±13)NH2 and [F/ G]NC(1±13)NH2 modi®cations) were also [Nphe 1]NC(1± 13)NH2 sensitive. Additionally, [Nphe 1]NC(1±13)NH2 also antagonized electrically stimulated contractions in mouse vas deferens, rat vas deferens and guinea pig ileum with pA2 values ranging between 5.96 and 6.65. Moreover, in vivo, [Nphe 1]NC(1±13)NH2 blocked the pronociceptive and antiopioid effects of intracerebroventriculary administered NC in the mouse tail withdrawal test and produced a dose-dependent antinociceptive action alone [7]. Collectively these data clearly demonstrate that [Nphe 1]NC(1± 13)NH2 is a selective antagonist for the NCR found both in the periphery and in the central nervous system. Two very recent publications corroborate this statement where NC dependent contraction of the mouse colon [22] and NC stimulated food intake in the rat [21] were also antagonised in a competitive fashion by [Nphe 1]NC(1±13)NH2. However, this compound is relatively weak and displays some residual agonist activity (max inhibition ,15%). However, we know that in this assay residual agonist activity is overestimated compared with recombinant systems expressing lower receptor densities or native receptors expressed in isolated tissues. For example [20], the nociceptin receptor ligand [F/G]NC(1±13)NH2 displays both antagonist (rat vas deferens, low levels of expression) and full agonist activity in CHONCR (high levels of expression) depending on the levels of expression (see below). [F/G]NC(1±13)NH2 (NC 1±13 template with a pseudo peptide bond between Phe 1 and Gly 2) was initially reported
Y. Hashimoto et al. / Neuroscience Letters 278 (2000) 109±112
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Fig. 1. Forskolin stimulated cAMP accumulation in CHONCR cells in the absence and presence of 10 mM [Nphe 1]NC(1±13)NH2. Concentration response curves to (A) nociceptin, (B) NC(1±13)NH2 (C) [F/G]NC(1-17)NH2, (D) [F/G]NC(1±13)NH2, (E) CAM6369 and (F) buprenorphine. Points indicate the mean and the vertical line the standard error of mean of ®ve to six experiments. Numerical values obtained from these data are represented in Table 1.
as a NC receptor antagonist in peripheral preparations [14]. [F/G]NC(1±13)NH2 was shown to prevent inhibition by NC of electrically-induced contraction of the mouse and rat vas deferens, the guinea pig ileum, and the renal pelvis [4]. However [F/G]NC(1±13)NH2 was later shown to be a full agonist in the central nervous system and in the cells transfected with NC receptors [5,8,9,20,26]. We have suggested that [F/G]NC(1±13)NH2 is in fact a low ef®cacy agonist. Indeed, this has recently been shown by Chiou et al. [10] where the pseudopeptide acted as a partial agonist for actiTable 1 Inhibition of cAMP accumulation in CHONCR cells in the absence or presence of 10 mM of [Nphe 1]NC(1±13)NH2 (Nphe 1) and estimated pA2 values a [Nphe 1]
Emax (%)
pEC50
pA2
NC
2 1
102.3 ^ 0.6 9.64 ^ 0.07 108.7 ^ 1.4 8.38 ^ 0.06 6.23 ^ 0.09
NC(1±13)NH2
2 1
103.7 ^ 1.1 9.79 ^ 0.08 103.9 ^ 1.1 8.51 ^ 0.05 6.12 ^ 0.12
[F/G]NC(1-17)NH2
2 1
101.5 ^ 0.6 9.14 ^ 0.08 103.0 ^ 0.6 7.76 ^ 0.06 6.36 ^ 0.10
[F/G]NC(1±13)NH2
2 1
101.5 ^ 0.6 8.53 ^ 0.06 108.8 ^ 2.1 7.03 ^ 0.11 6.47 ^ 0.16
CAM6369
2 1
103.8 ^ 0.4 9.18 ^ 0.02 109.1 ^ 1.6 7.68 ^ 0.17 6.48 ^ 0.18
Buprenorphine
2 1
95.2 ^ 1.1 7.40 ^ 0.04 100.6 ^ 0.4 6.03 ^ 0.16 6.35 ^ 0.18
a
Data are mean ^ SEM (n 5±6).
vation of inwardly rectifying K 1 channels in the rat periaqueductal gray. [F/G]NC(1±17)NH2 (i.e. the full NC peptide with a pseudo peptide bond between Phe 1 and Gly 2) acts as an antagonist in the periphery (mouse vas deferens) with a pA2 value of 7.0 [23] and as anticipated a full agonist for inhibition of cAMP formation in CHONCR cells in the present study. The hexapeptide CAM6369 was identi®ed as part of a large haxapeptide screen by Dooley et al. [13] and has been reported to be a partial agonist for stimulation of [ 35S]-GTPg S binding and inhibition of cAMP accumulation in CHO cells transfected with NCR. Berger et al. [2] reported that CAM6369 antagonized the stimulation of [ 35S]-GTPg S binding to G-proteins by NC in concentration-dependent manner in membranes and slices of rat brain. Moreover the same group also reported that this compound acted as a full agonist in the mouse in vivo [3]. In the present study CAM6369 acted as full agonist. This is not entirely unexpected as CHONCR cells express high levels of NCR. More importantly the effects of CAM6369 were competitively antagonized by [Nphe 1]NC(1±13)NH2. Buprenorphine is a clinically used opioid with af®nity for m and k -opioid receptors [25]. However, Wnendt et al. [25] have recently reported that buprenorphine was a full agonist at the NCR using a reporter gene assay with a pIC50 of 8.1. This compares favourably with the value of 7.4 reported here. The activity of this non-peptide agonist was also reversed by [Nphe 1]NC(1±13)NH2. In conclusion, we demonstrate that [Nphe 1]NC(1± 13)NH2 competitively antagonizes the action of a battery of NCR agonists encompassing both peptide and nonpeptide structures.
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We would like to thank Dr. F. Marshall and Mrs. N. Bevan of Glaxo±Wellcome, Stevenage, Herts, UK for providing CHO cells expressing the human NC receptor. [1] Bauer, M., Nakazi, M., Kathmann, M., Gothert, J. and Schlicker, E, The stereoselective kappa opioid receptor antagonist Mr 2266 does not exhibit stereoselectivity as an antagonist at the orphan opioid (ORL1)receptor. Naunyn Schmiedeberg's Arch. Pharmacol., 359 (1999) 17±20. [2] Berger, H., Albrecht, E., Wallukat, G. and Bienert, M., Antagonism by acetyl-RYYRIK-NH2 of G protein activation in rat brain preparations and of chronotropic effect on rat cardiomyocytes evoked by nociceptin/orphanin FQ. Br. J. Pharmacol., 126 (1999) 555±558. [3] Berger, H., Albrecht, E., Wallukat, G., Calo', G., Bigoni, R. and Bienert, M., Ac-RYYRIK-NH2, a nociceptin/orphanin FQ receptor ligand, exhibits properties of an antagonist and agonist as well. Regul. Pept., 80 (1999) 122. [4] Bigoni, R., Giuliani, S., Calo', G., Rizzi, A., Guerrini, R., Salvadori, S., Regoli, D. and Maggi, C.A., Characterization of nociceptin receptors in the periphery: in vitro and in vivo studies. Naunyn Schmiedeberg's Arch. Pharmacol., 359 (1999) 160±167. [5] Butour, J.-L., Moisand, C., Mollereau, C. and Meunier, J.-C., [Phe 1c(CH2-NH)Gly 2]nociceptin(1±13)-NH2 is an agonist of the nociceptin (ORL1) receptor. Eur. J. Pharmacol., 349 (1998) R5±R6. [6] Calo', G., Guerrini, R., Bigoni, R., Rizzi, A., Bianchi, C., Regoli, D. and Salvadori, S., Structure-activity study of the nociceptin(1±13)-NH2 N-terminal tetrapeptide and discovery of a nociceptin receptor antagonist. J. Med. Chem., 41 (1998) 3360±3366. [7] Calo', G., Guerrini, R., Bigoni, R., Rizzi, A., Marzola, G., Okawa, H., Bianchi, C., Lambert, D.G., Salvadori, S. and Regoli, D., Characterization of [Nphe 1]nociceptin(1± 13)NH2, a novel selective nociceptin receptor antagonist, Proceeding of the International Narcotics Research Conference Meeting, S1-5, Saratoga Springs, New York, July 10± 15 (1999). [8] Calo, G., Rizzi, A., Marzola, G., Guerrini, R., Salvadori, S., Beani, L., Regoli, D. and Bianchi, C., Pharmacological characterization of the nociceptin receptor mediating hyperalgesia in the mouse tail withdrawal assay. Br. J. Pharmacol., 125 (1998) 373±378. [9] Carpenter, K.J. and Dickenson, A.H., Evidence that [Phe 1c(CH2-NH)Gly 2]nociceptin-(1±13)-NH2, a peripheral ORL-1 receptor antagonist, acts as an agonist in the rat spinal cord. Br. J. Pharmacol., 125 (1998) 949±951. [10] Chiou, L.-C., [Phe 1c(CH2-NH)Gly 2]nociceptin-(1±13)-NH2 activation of an inward recti®er as a partial agonist of ORL1 receptors in rat periaqueductal gray. Br. J. Pharmacol., 128 (1999) 103±107. [11] Connor, M., Vaughan, C.W., Chieng, B. and Christie, M.J., Nociceptin receptor coupling to a potassium conductance in rat locus coeruleus neurons in vitro. Br. J. Pharmacol., 119 (1996) 1614±1618. [12] Connor, M., Yeo, A. and Henderson, G., The effect of nociceptin on Ca 21 channel current and intracellular Ca 21 in the SH-SY5Y human neuroblastoma cell line. Br. J. Pharmacol., 118 (1996) 205±207.
[13] Dooley, C.T., Spaeth, C.G., Berzeteigurske, I.P., Craymer, K., Adapa, I.D., Brandt, S.R., Houghten, R.A. and Toll, L., Binding and in vitro activities of peptides with high af®nity for the nociceptin/orphanin FQ receptor, ORL1. J. Pharmacol. Exp. Ther., 283 (1997) 735±741. [14] Guerrini, R., Calo', G., Rizzi, A., Bigoni, R., Bianchi, C., Salvadori, S. and Regoli, D., A new selective antagonist of the nociceptin receptor. Br. J. Pharmacol., 123 (1998) 163±165. [15] Kno¯ach, F., Reinscheid, R.K., Civelli, O. and Kemp, J.A., Modulation of voltage-gated calcium channels by orphanin FQ in freshly dissociated hippocampal neurons. J. Neurosci., 16 (1996) 6657±6664. [16] Kobayashi, T., Ikedae, K., Togashi, S., Itoh, N. and Kumanishi, T., Effects of (ligands on the nociceptin/orphanin FQ receptor co-expressed with G-protein-activated K 1 channel in Xenopus oocytes. Br. J. Pharmacol., 120 (1997) 986±987. [17] Meunier, J.-C., Nociceptin /orphanin FQ and the opioid receptor like ORL1 receptor. Eur. J. Pharmacol., 340 (1997) 1±15. [18] Noda, Y., Mamiya, T., Nabeshima, T., Nishi, M., Higashioka, M. and Takeshima, H., Loss of antinociception induced by naloxone benzoylhydrazone in nociceptin receptor-knockout mice. J. Biol. Chem., 273 (1998) 18047±18051. [19] Okawa, H., Hirst, R.A., Smart, D., McKnight, A.T. and Lambert, D.G., Studies on the coupling of recombinant ORL1 receptors to adenylyl cyclase. Br. J. Pharmacol., 123 (1998) 218. [20] Okawa, H., Nicol, B., Bigoni, R., Hirst, R.A., Calo, G., Guerrini, R., Rowbotham, D.J., Smart, D., McKnight, A.T. and Lambert, D.G., Comparison of the effects of [Phe 1c(CH2NH)Gly 2]nociceptin(1±13)NH2 in the rat brain, rat vas deferens, and CHO cells expressing recombinant human nociceptin receptors. Br. J. Pharmacol., 127 (1999) 123±130. [21] Polidori, C., Calo, G., Ciccocioppo, R., Guerrini, R., Regoli, D. and Massi, M., Pharmacological characterization of the nociceptin receptor mediating hyperphagia: identi®cation of a selective antagonist. Psychopharmacology, (1999) in press. [22] Rizzi, A., Bigoni, R., Calo, G., Guerrini, R., Salvadori, S. and Regoli, D., [Nphe 1]nociceptin(1±13)NH2 antagonizes nociceptin effects in the mouse colon. Eur. J. Pharmacol., (1999) in press. [23] Salvadori, S., Guerrini, R., Calo', G. and Regoli, D., Structure activity studies on nociceptin/orphanin FQ: from full agonist, to partial agonist, to pure antagonist. Il Farmaco, (1999) in press. [24] Vaughan, C.W., Ingram, S.L. and Christie, M.J., Action of ORL1 receptor ligand nociceptin on membrane properties of rat periaqueductal gray neurons in vitro. J. Neurosci., 17 (1997) 996±1003. [25] Wnendt, S., Kruger, T., Janocha, E., Hildebrandt, D. and Englberger, W., Agonistic effect of buprenorphine in a Nociceptin/OFQ receptor-triggered reporter gene assay. Mol. Pharmacol., 56 (1999) 334±338. [26] Xu, I.S., Wiesenfeld-Hallin, Z. and Xu, X.J., [Phe 1c(CH2NH)Gly 2]-nociceptin-(1±13)NH2, a proposed antagonist of the nociceptin receptor, is a potent and stable agonist in the rat spinal cord. Neurosci. Lett., 249 (1998) 127± 130. [27] Zaki, P.A. and Evans, C.J., ORL-1: an Awkward child of the opioid receptor family. Neuroscientist, 4 (1998) 172±184.
Antagonistic effects of [Nphe 1]nociceptin(1±13)NH2 on nociceptin receptor mediated inhibition of cAMP formation in Chinese hamster ovary cells stably expressing the recombinant human nociceptin receptor Y. Hashimoto a, G. Calo' b, R. Guerrini c, G. Smith a, D.G. Lambert a,* a
University Department of Anaesthesia and Pain Management, Leicester Royal In®rmary, Leicester, LE1 5WW, UK Department of Experimental and Clinical Medicine, Section of Pharmacology, University of Ferrara, via Fossato di Mortara, 17, 44100 Ferrara, Italy c Department of Pharmaceutical Sciences, University of Ferrara, via Fossato di Mortara, 17, 44100 Ferrara, Italy
b
Received 20 September 1999; received in revised form 15 November 1999; accepted 15 November 1999
Abstract Nociceptin/orphanin FQ (NC) is the endogenous ligand for the nociceptin receptor (NCR) which is negatively coupled to adenylyl cyclase to inhibit the formation of cAMP. In this study we describe the inhibitory action of the novel NC analogue, [Nphe 1]nociceptin(1±13)NH2 on cAMP formation in Chinese hamster ovary cells expressing the human NCR. NC, NC(1±13)NH2, the pseudopeptides [Phe 1c(CH2±NH)Gly 2]NC(1±17)NH2 and [Phe 1c(CH2±NH)Gly 2]NC(1±13)NH2, the hexapeptide, acetyl-Arg-Tyr-Tyr-Arg-Trp-Lys-NH2 and buprenorphine all produced a concentration dependent inhibition of forskolin stimulated cAMP formation. This inhibition was competitively reversed by [Nphe 1]NC(1±13)NH2 with essentially identical pA2 values (6.12±6.48). [Nphe 1]NC(1±13)NH2 showed per se a negligible residual agonist activity (a , 0.15). q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nociceptin/orphanin FQ; Antagonist; [Nphe1]nociceptin(1±13)NH2; cAMP
Nociceptin/orphanin FQ (NC) is the endogenous ligand for the nociceptin receptor (NCR) [17,27]. NCR is coupled via the inhibitory guanine nucleotide binding protein (Gprotein) Gi/o to adenylyl cyclase [17,27], potassium channels [11,24], and calcium channels [12,15]. NCR activation leads to reduced cAMP formation, an enhanced outward K 1 conductance and closing of voltage sensitive Ca 21 channels. Whilst there is reasonable structural homology between opioid and NCR and similarity in the transduction pathways employed the physiological roles of these systems differ. Opioids produce classic antinociceptive responses. NC produces hyperalgesia or anti-opioid actions following intracerebroventricular injection and antinociception when administered intrathecally [17,27]. Thus, the precise role of NC in nociception remains to be resolved. There is currently a paucity of compounds active at the NCR and the lack of a selective, competitive antagonist has * Corresponding author. Tel.: 144-116-258-5291; fax: 144-116285-4487. E-mail address: [email protected] (D.G. Lambert)
clearly hampered the characterization of the NC/NCR system. Ligands active at NCR include naloxone benzoylhydrazone [18], rimcazole and carbamapentane [16] and Mr2266 [1]. All are relatively weak and do not display selectivity for NCR. We have recently reported the activity of the pseudopeptide [Phe 1c(CH2±NH)Gly 2]Nociceptin(1±13)NH2 ([F/ G]NC(1±13)NH2) in the guinea pig ileum and mouse vas deferens where an antagonist action at the NCR was observed [14]. However, we and others have also shown that [F/G]NC(1±13)NH2 inhibited forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells (CHO) stably expressing the nociceptin receptor [5,20,25] and inhibited K 1 evoked-glutamate release from rat cerebrocortical slices [20] indicating an agonist action. [F/ G]NC(1±13)NH2 is clearly of limited value in the elucidation of the role of NC/NC receptor system in the central nervous system. Earlier this year we reported to the International Narcotics Research Conference meeting the characteristics of a novel NC analogue, [Nphe 1]nociceptin(1±13)NH2
0304-3940/99/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 91 5- 5
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([Nphe 1]NC(1±13)NH2) in a variety of preparations and have noted a (weak) competitive antagonist pro®le, essentially devoid of any agonist activity and with good selectivity for NCR over classical m, d and k opioid receptors [7]. In this study we have further characterized the antagonistic activity of [Nphe 1]NC(1±13)NH2 against a battery of NCR ligands; NC, NC(1±13)NH2, [F/G]NC(1±17)NH2, [F/ G]NC(1±13)NH2 [6,14,20,23], CAM6369 (acetyl-Arg-TyrTyr-Arg-Trp-Lys-NH2 [13,19]), and buprenorphine [25]. The chemicals and their sources were as follows; NC, NC(1±13)NH2, [F/G]NC(1±17)NH2, [F/G]NC(1±13)NH2 and [Nphe 1]NC(1±13)NH2 were synthesized at one of our institutes as previously described [6,23]. CAM6369 was a gift from Drs. D. Smart and A.T. McKnight (Parke±Davis Neuroscience Research Centre, Cambridge, UK). Cell culture medium (Dulbecco's Modi®ed Eagles medium (DMEM) and Hams F12), foetal calf serum, hygromycin B, and G418 were from Gibco (Paisley, UK). [2,8± 3H]cAMP (28.4 Ci/mmol) was purchased from NEN DuPont (Boston, MA, USA). HEPES was from USB (Cleveland, OH, USA). All other reagents were from Sigma Chemical Co. (Poole, UK). CHO cells stably expressing the human nociceptin receptor, CHONCR (transfected using pCIN5), were obtained from Dr. F. Marshall and Mrs. N. Bevan of Glaxo±Wellcome, Stevenage, Hearts, UK. CHONCR cells expressing 1.8 pmol/mg protein NCR [20] were cultured in DMEM/Hams F12 medium (v/v 50:50) supplemented with foetal bovine serum 5% (v/v), G418 200 mg/ml, and hygromycin B 200 mg/ml at 378C in 5% CO2/humidi®ed air. Cultures were passaged twice weekly and used for investigation when con¯uent (3±4 days). Cells were harvested with 10 mM HEPES buffered saline/0.05% EDTA, pH 7.4, washed twice with and resuspended in Krebs/HEPES buffer of the following composition; (mM): Na 1 (143.3), K 1 (4.7), Ca 21 (2.15), Mg 21 (1.2), Cl 2 (125.6), H2PO422 (1.2), SO422 (1.2), Glucose (11.7), and HEPES (10), BSA 0.5%, pH 7.4 with 10 M NaOH. cAMP formation was measured in 0.3 ml volumes of whole cell suspensions in the presence of isobutylmethylxanthine (IBMX; 1 mM) and forskolin (FSK: 1 mM). Peptidase inhibitors were not used as these did not in¯uence the response to NC (data not shown). NCR agonists were included in various concentrations in order to obtain a full concentration response curve. In some experiments 10mM [Nphe 1]NC(1±13)NH2 was included. After 15 min of incubation at 378C, reactions were terminated and cAMP extracted by the addition of HCl (10 M). The reaction mixture was neutralized with NaOH (10 M) and Tris±HCl (1 M, pH 7.4). cAMP mass was measured in the supernatants using a protein binding assay as described by Okawa et al. [20]. All data are expressed as mean ^ SEM. The concentration of each agonist producing half maximal inhibition of cAMP formation (EC50) was obtained by computer-assisted curve ®tting of individual curves. All curve ®tting was performed using PRISM-V2.0 (GraphPad software, San
Diego, CA, USA). Antagonist af®nities are expressed in terms of pA2, which is the negative logarithm to base 10 of the antagonist molar concentration that makes it necessary to double the agonist concentration to elicit the original submaximal response. pA2 was calculated using the following formula: pA2 2log10 CR-1=antagonist, where CR is the ratio of EC50 in the presence and absence of antagonist. A slope of unity of the Schild regression line against NC has been reported previously [7] and is therefore assumed for the additional agonists examined here. All agents examined inhibited forskolin-stimulated cAMP accumulation in CHONCR cells in a concentrationdependent fashion with maximal inhibition of around 100% (Fig. 1A±F). [Nphe 1]NC(1±13)NH2 produced a competitive antagonism of each agonist response resulting in a parallel rightward shift of the concentration response curve. Maximal inhibition and pEC50 values in the absence and presence of [Nphe 1]NC(1±13)NH2 along with the estimated pA2 values are presented in Table 1. There was a small direct inhibition of cAMP formation in response to [Nphe 1]NC(1±13)NH2 alone (14.4 ^ 0.8%) which did not differ from our previous report [7]. We report that [Nphe 1]NC(1±13)NH2 acts as a competitive antagonist against a range of NCR agonists with essentially identical pA2 values. The pA2 against NC inhibition of cAMP formation is in agreement with the value reported by us previously [7]. Moreover, the effects of NC(1±13)NH2 (the template used for both [Nphe 1]NC(1±13)NH2 and [F/ G]NC(1±13)NH2 modi®cations) were also [Nphe 1]NC(1± 13)NH2 sensitive. Additionally, [Nphe 1]NC(1±13)NH2 also antagonized electrically stimulated contractions in mouse vas deferens, rat vas deferens and guinea pig ileum with pA2 values ranging between 5.96 and 6.65. Moreover, in vivo, [Nphe 1]NC(1±13)NH2 blocked the pronociceptive and antiopioid effects of intracerebroventriculary administered NC in the mouse tail withdrawal test and produced a dose-dependent antinociceptive action alone [7]. Collectively these data clearly demonstrate that [Nphe 1]NC(1± 13)NH2 is a selective antagonist for the NCR found both in the periphery and in the central nervous system. Two very recent publications corroborate this statement where NC dependent contraction of the mouse colon [22] and NC stimulated food intake in the rat [21] were also antagonised in a competitive fashion by [Nphe 1]NC(1±13)NH2. However, this compound is relatively weak and displays some residual agonist activity (max inhibition ,15%). However, we know that in this assay residual agonist activity is overestimated compared with recombinant systems expressing lower receptor densities or native receptors expressed in isolated tissues. For example [20], the nociceptin receptor ligand [F/G]NC(1±13)NH2 displays both antagonist (rat vas deferens, low levels of expression) and full agonist activity in CHONCR (high levels of expression) depending on the levels of expression (see below). [F/G]NC(1±13)NH2 (NC 1±13 template with a pseudo peptide bond between Phe 1 and Gly 2) was initially reported
Y. Hashimoto et al. / Neuroscience Letters 278 (2000) 109±112
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Fig. 1. Forskolin stimulated cAMP accumulation in CHONCR cells in the absence and presence of 10 mM [Nphe 1]NC(1±13)NH2. Concentration response curves to (A) nociceptin, (B) NC(1±13)NH2 (C) [F/G]NC(1-17)NH2, (D) [F/G]NC(1±13)NH2, (E) CAM6369 and (F) buprenorphine. Points indicate the mean and the vertical line the standard error of mean of ®ve to six experiments. Numerical values obtained from these data are represented in Table 1.
as a NC receptor antagonist in peripheral preparations [14]. [F/G]NC(1±13)NH2 was shown to prevent inhibition by NC of electrically-induced contraction of the mouse and rat vas deferens, the guinea pig ileum, and the renal pelvis [4]. However [F/G]NC(1±13)NH2 was later shown to be a full agonist in the central nervous system and in the cells transfected with NC receptors [5,8,9,20,26]. We have suggested that [F/G]NC(1±13)NH2 is in fact a low ef®cacy agonist. Indeed, this has recently been shown by Chiou et al. [10] where the pseudopeptide acted as a partial agonist for actiTable 1 Inhibition of cAMP accumulation in CHONCR cells in the absence or presence of 10 mM of [Nphe 1]NC(1±13)NH2 (Nphe 1) and estimated pA2 values a [Nphe 1]
Emax (%)
pEC50
pA2
NC
2 1
102.3 ^ 0.6 9.64 ^ 0.07 108.7 ^ 1.4 8.38 ^ 0.06 6.23 ^ 0.09
NC(1±13)NH2
2 1
103.7 ^ 1.1 9.79 ^ 0.08 103.9 ^ 1.1 8.51 ^ 0.05 6.12 ^ 0.12
[F/G]NC(1-17)NH2
2 1
101.5 ^ 0.6 9.14 ^ 0.08 103.0 ^ 0.6 7.76 ^ 0.06 6.36 ^ 0.10
[F/G]NC(1±13)NH2
2 1
101.5 ^ 0.6 8.53 ^ 0.06 108.8 ^ 2.1 7.03 ^ 0.11 6.47 ^ 0.16
CAM6369
2 1
103.8 ^ 0.4 9.18 ^ 0.02 109.1 ^ 1.6 7.68 ^ 0.17 6.48 ^ 0.18
Buprenorphine
2 1
95.2 ^ 1.1 7.40 ^ 0.04 100.6 ^ 0.4 6.03 ^ 0.16 6.35 ^ 0.18
a
Data are mean ^ SEM (n 5±6).
vation of inwardly rectifying K 1 channels in the rat periaqueductal gray. [F/G]NC(1±17)NH2 (i.e. the full NC peptide with a pseudo peptide bond between Phe 1 and Gly 2) acts as an antagonist in the periphery (mouse vas deferens) with a pA2 value of 7.0 [23] and as anticipated a full agonist for inhibition of cAMP formation in CHONCR cells in the present study. The hexapeptide CAM6369 was identi®ed as part of a large haxapeptide screen by Dooley et al. [13] and has been reported to be a partial agonist for stimulation of [ 35S]-GTPg S binding and inhibition of cAMP accumulation in CHO cells transfected with NCR. Berger et al. [2] reported that CAM6369 antagonized the stimulation of [ 35S]-GTPg S binding to G-proteins by NC in concentration-dependent manner in membranes and slices of rat brain. Moreover the same group also reported that this compound acted as a full agonist in the mouse in vivo [3]. In the present study CAM6369 acted as full agonist. This is not entirely unexpected as CHONCR cells express high levels of NCR. More importantly the effects of CAM6369 were competitively antagonized by [Nphe 1]NC(1±13)NH2. Buprenorphine is a clinically used opioid with af®nity for m and k -opioid receptors [25]. However, Wnendt et al. [25] have recently reported that buprenorphine was a full agonist at the NCR using a reporter gene assay with a pIC50 of 8.1. This compares favourably with the value of 7.4 reported here. The activity of this non-peptide agonist was also reversed by [Nphe 1]NC(1±13)NH2. In conclusion, we demonstrate that [Nphe 1]NC(1± 13)NH2 competitively antagonizes the action of a battery of NCR agonists encompassing both peptide and nonpeptide structures.
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