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orphanin FQ inhibits glutamate release from rat cerebellar and brain stem slices
Neuroscience Letters 326 (2002) 85–88 www.elsevier.com/locate/neulet
Nociceptin/orphanin FQ inhibits glutamate release from rat cerebellar and brain stem slices B. Nicol 1, D.J. Rowbotham, D.G. Lambert* University Department of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester LE1 5WW, UK Received 8 February 2002; received in revised form 12 March 2002; accepted 18 March 2002
Abstract We have previously demonstrated that nociceptin/orphanin FQ (N/OFQ), inhibits K 1 depolarisation-evoked glutamate release from rat cerebrocortical slices. In this study we have examined the effects of N/OFQ on glutamate release from rat cerebellar and brain stem slices as there are regional differences in nociceptin/orphanin FQ receptor (NOP) expression. Slices were depolarised with two pulses of 46 mM K 1 (S1 and S2) with N/OFQ added after S1. Glutamate (non-radioactive) was measured using a fluorescence-based assay. N/OFQ effects were assessed by measuring area under S1 and S2 release curves and calculation of S2/S1 ratios. In cerebellar slices K 1 evoked S2/S1 ratio was 1.17 ^ 0.10 (n ¼ 28). This was reduced in a concentration dependent (EC50 22 nM; Emax 46%) and naloxone (10 mM) insensitive manner by N/OFQ. In the brain stem K 1 evoked glutamate release was considerably reduced compared to cerebellum. In several preparations K 1 failed to evoke a significant release. In those that did K 1 evoked S2/S1 ratio was 1.03 ^ 0.07 (n ¼ 13). A total of 100 nM N/OFQ reduced this by 38 ^ 12% and this response was naloxone insensitive. Due to this small response and its variability we could not construct a full concentration response curve. In conclusion we have demonstrated a functional NOP in rat cerebellum and brain stem that inhibits the release of glutamate. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Glutamate release; Nociceptin/orphanin FQ; Cerebellar slice; Brain stem slice
Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the orphan opioid receptor-like receptor (NOP) [7,16]. This terminology is consistent with recent suggestions by IUPHAR [4]. N/OFQ is involved in many central (and peripheral) responses including pain processing, anxiolysis, feeding, motor and cardiovascular control [3,8]. NOP receptors are widely distributed in both tissues derived from the central nervous system; including cerebrocortex, cerebellum and brain stem [5,9] and a range of peripheral tissues including intestine, skeletal muscle, vas deferens, spleen and lymphocytes [1,15,20,21]. Indeed, in a series of radioligand binding studies to crude membranes prepared from rat cerebrocortes, cerebellum and brain stem using [ 125I]Y 14N/OFQ as a radioligand we reported expression of 180, 12 and 52 fmol/mg protein of NOP, respectively [13]. Very low levels of N/OFQ expression in the cerebellum are consistent with the literature [9] and it is well known * Corresponding author. Tel.: 144-116-258-5291; fax: 144-116285-4487. E-mail address: [email protected] (D.G. Lambert). 1 Present address: Animal Health Discovery, Pfizer, Sandwich, Kent, UK.
that this brain region is particularly rich in glutamatergic neurones, e.g. purkinje fibres and N/OFQ has effects on motor control (see refs [3,8]). In the brain stem there are several nuclei that express NOP including locus coeruleus, raphe magnus, nucleus ambiguous, dorsal motor nucleus of the vagus (DMNV) and nucleus of the tractus solitarii (NTS) (see ref. [9]). In both DMNV and NTS, involved in cardiovascular control, there is evidence for a role for glutamatergic transmission [18,19]. We have previously demonstrated that N/OFQ, N/ OFQ(1–13)NH2 and the partial agonist [F/G]N/OFQ(1– 13)NH2 inhibit the release of glutamate from rat cerebrocortical slices. The response to N/OFQ was insensitive to the opioid antagonist naloxone [11,14]. In addition we have also recently demonstrated that N/OFQ inhibits ischaemia induced glutamate release in the same preparation [10]. Moreover, N/OFQ has been shown to inhibit the release of a wide range of other neurotransmitters including noradrenaline, dopamine, serotonin and acetylcholine [17]. In this study we have examined the effects of N/OFQ on K 1-evoked release of endogenous glutamate from rat cerebellar and brain stem slices and compared these data to our
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Fig. 1. Potassium (46 mM) evoked glutamate release from rat cerebellar slices is extracellular Ca 21 dependent and inhibited by nociceptin (N/OFQ). In panels A and B two 2 min pulses of 46 mM K 1 (S1 and S2) were applied to the slices resulting in monophasic releases of glutamate which are expressed relative to the mean of the first three fractions collected. In panel A immediately after S1 the buffer was replaced with nominally Ca 21 free solution containing 0.1 mM EGTA until the end of the experiment. In panel B immediately after S1 the buffer was replaced with solution containing 100 nM N/OFQ until the end of the experiment. In panel C, based on S2/S1 ratios, N/OFQ produced a concentration dependent inhibition of evoked release (whole curve P , 0:05) with an estimated EC50 of 22 nM. In panel D the response to 100 nM N/OFQ was insensitive to 10 mM naloxone (*P , 0:05 compared to control). Data are mean ^ SEM (n . 5).
previous study in the cerebrocortex as there are regional differences in NOP expression. Effects of N/OFQ on glutamate release from rat brain slices were examined essentially as described previously [11,14]. Briefly, the brain was removed from female Wistar rats (200–250 g) and the cerebellum and brain stem dissected. Typically tissue from two animals was pooled, this was particularly important for the brain stem due to its small size. Slices were cut separately and washed three times in 95% O2 and 5% CO2 Krebs buffer, pH 7.4, prior to agitation in a shaking water bath at 378C for 40 min. A total of 1 ml of gravity packed slices were pipetted into a perfusion chamber. Slices were perfused at 378C for 60 min at 1 ml/min prior to collection of 2 min fractions. A 2 min pulse of 46 mM K 1 (Na 1 adjusted) was applied (S1) following 6 min of perfusion. Slices were perfused for a further 30 min, prior to the second application of a 2 min pulse of 46
mM K 1 (S2). Perfusate glutamate concentrations were measured fluorimetrically as described previously [11,12,14] and expressed relative to the mean of the first three basal samples. Based on the raw data, area under the S1 and S2 release profiles and an S2/S1 ratio was calculated. Use of S2/S1 ratios allows for internal control of a variable S1 phase between tissues prepared from different animals and on different days. To examine the effects of N/OFQ on glutamate release, N/OFQ (in the presence of 30 mM amastatin, bestatin, captopril and phosphoramidon) was applied immediately after S1 until the end of the experiment (also present during S2). Agents inhibiting release will reduce S2 and hence the S2/S1 ratio. As it was not possible to construct a full concentration response curve in a single animal three conditions were tested in each experiment; control, high concentration of N/OFQ and low concentration of N/OFQ in random order. In some experiments, naloxone (10 mM)
B. Nicol et al. / Neuroscience Letters 326 (2002) 85–88
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Fig. 2. Potassium (46 mM) evoked glutamate release from rat brain stem slices is inhibited by nociceptin (N/OFQ). In panel A two 2 min pulses of 46 mM K 1 (S1 and S2) were applied to the slices resulting in monophasic releases of glutamate which are expressed relative to the mean of the first three fractions collected. This was inhibited by 100 nM N/OFQ applied immediately after S1 until the end of the experiment. In panel B the response to 100 nM N/OFQ was insensitive to 10 mM naloxone (*P , 0:05 compared to control). Data are mean ^ SEM (n . 5).
and Ca 21 free buffer plus 0.1 mM EGTA were used to block classical (m, d and k) opioid receptors and confirm Ca 21 sensitive release respectively. N/OFQ was provided by Dr R. Guerrini of the University of Ferrara, Italy. Phosphoramidon was from peptide institute (Osaka, Japan). All other reagents were from Sigma Chemical Co. (Poole, UK). Data are presented as the mean ^ SEM (n, independent experiments). The pharmacological terminology adopted in this study is in line with the IUPHAR recommendations [6]: EC50 is concentration of an agonist that produces 50% of the maximum possible effect, Emax. All curve fitting was performed using GRAPHPAD-PRISM. Statistical comparisons of paired samples were made using Freidman’s analysis of variance followed by Dunn’s test or Wilcoxon Rank sum test as appropriate and considered significant when P , 0:05. A 2 min pulse of 46 mM K 1 produced a monophasic release of glutamate from rat cerebellar slices reaching some 2.5-fold over basal. A second pulse of K 1 also produced a similar release profile (Figs. 1A,B). In common with the cerebrocortex [12] K 1 evoked glutamate release was substantially extracellular Ca 21 dependent as removal of extracellular Ca 21 and addition of 0.1 mM EGTA reduced the S2/S1 ratio by some 70% compared to control (80% in cerebrocortex [12]) (Fig. 1A). In 28 control experiments in this tissue S2/S1 ratios were 1.17 ^ 0.10. When N/ OFQ was included prior to S2 the S2 release was reduced. An example at 100 nM N/OFQ is shown in Fig. 1B where a 40 ^ 9% (n ¼ 7) inhibition of release was observed. The effects of N/OFQ were concentration dependent with Emax and EC50 values of 46% and 22 nM, respectively (Fig. 1C). In the rat cerebrocortex we reported EC50 and Emax values for N/OFQ of 51 nM and 68%, respectively [11]. The N/ OFQ (100 nM) response was not due to activation of neuro-
nal opioid receptors as this was naloxone (10 mM) insensitive (Fig. 1D). In addition we were unable to detect any change in basal glutamate release immediately following N/OFQ application (data not shown). In the brain stem K 1 evoked glutamate release was considerably reduced compared to the cerebellum. In contrast to cerebellar slices in several preparations K 1 failed to evoke a significant release. In those (n ¼ 13) that did respond, we were able to detect monophasic release profiles in response to K 1 challenge during both S1 and S2 yielding an S2/S1 ratio of 1.03 ^ 0.07 (Fig. 2A). When N/OFQ was included prior to S2 the S2 release was reduced. An example at 100 nM N/OFQ is shown in Fig. 2A where a 38 ^ 12% (n ¼ 5) inhibition of release was observed, this was not significantly different (P . 0:05) from that observed at 100 nM in the cerebellum. In view of the small response and its variability we did not construct a full concentration response curve. At 100 nM N/OFQ, naloxone (10 mM) was ineffective (Fig. 2B). In this study we have clearly demonstrated that the peptide N/OFQ inhibits the release of glutamate from rat cerebellar and brain stem slices. This was mediated by the NOP as the response was insensitive to naloxone. Whilst it would have been preferable to examine the effects of the selective NOP antagonist [Nphe 1]N/OFQ(1–13)NH2 [2] this was not available to us at the time these studies were undertaken. In the cerebellum we were able to construct a full concentration response curve that yielded EC50 values similar to that previously reported in rat cerebrocortex [11]. However, in the brain stem such analysis was not possible. Reduced K 1-evoked glutamate release in the brain stem (and more specifically its absence in some preparations) compared to cerebellum or cerebrocortex [11] might suggest a high level of N/OFQ-ergic tone in this tissue
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such that endogenously released N/OFQ is sufficient to abolish evoked release. In view of the small degree of inhibition produced by addition of a relatively high concentration of exogenous N/OFQ this supposition is, in our view, unlikely. However, in the absence of studies utilising selective NOP antagonists (i.e. will pre-treatment with NOP antagonists increase the number of tissue preparations that respond and enhance the size of response in those that already do respond) this supposition cannot be completely ruled out. In the cortex we have previously demonstrated that N/ OFQ inhibits the release of glutamate in response to a depolarising challenge [11,14] in much the same way as m and k (but not d) opioids [12]. Coupled with the Ca 21 sensitivity, these data are in agreement with the results of the present study. In addition, we have also shown that N/OFQ inhibits ischaemia induced glutamate release and tentatively suggest that N/OFQ and the newer non-peptide agonists might possess neuroprotective properties. In conclusion we have demonstrated a functional NOP in rat cerebellum and brain stem that inhibits the release of glutamate. Further studies in the cerebellum with a range of non-peptide agonists and antagonists are warranted.
[8]
[9]
[10]
[11]
[12]
[13]
[14]
We are grateful to Dr R Guerrini (University of Ferrara, Italy) for providing N/OFQ. Funded by the Wellcome Trust. [1] Bigoni, R., Calo’, G., Guerrini, R., Strupish, J., Rowbotham, D.J. and Lambert, D.G., Effects of nociceptin and endomorphin 1 on the electrically stimulated human vas deferens, Br. J. Clin. Pharmacol., 51 (2001) 355–358. [2] Calo’, G., Guerrini, R., Bigoni, R., Rizzi, A., Marzola, G., Okawa, H., Bianchi, C., Lambert, D.G., Salvadori, S. and Regoli, D., Characterisation of [Nphe 1]nociceptin(1– 13)NH2, a new selective nociceptin receptor antagonist, Br. J. Pharmacol., 129 (2000) 1183–1193. [3] Calo’, G., Guerrini, R., Rizzi, A., Salvadori, S. and Regoli, D., Pharmacological characterisation of nociceptin and its receptor: a novel therapeutic target, Br. J. Pharmacol., 129 (2000) 1261–1283. [4] Cox, B.M., Chavkin, C., Christie, M.J., Civelli, O., Evans, C., Hamon, M.D., Hoellt, V., Kieffer, B., Kitchen, I., McKnight, A.T., Meunier, J.C. and Portoghese, P.S., Opioid receptors, In D. Girdlestone (Ed.), The IUPHAR Compendium of Receptor Characterisation and Classification, IUPHAR Media Ltd, London, 2000. [5] Darland, T. and Grandy, D.K., The orphanin FQ system: an emerging target for the management of pain?, Br. J. Anaesth., 81 (1998) 29–37. [6] Jenkinson, D.H., Barnard, E.A., Hoyer, D., Humphrey, P.P.A., Leff, P. and Shankley, N.P., International Union of Pharmacology Committee on receptor nomenclature and drug classification. XI Recommendations on terms and symbols in quantitative pharmacology, Pharmacol. Rev., 47 (1995) 255–266. [7] Meunier, J.C., Mollereau, C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P., Butour, J.L., Guillemot, J.C.,
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Ferrara, P., Monserrat, B., Mazarguil, H., Vassart, G., Parmentier, M. and Costentin, J., Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor, Nature, 377 (1995) 532–535. Mogil, J.S. and Pasternak, G.W., The molecular and behavioural pharmacology of the orphanin FQ/nociceptin peptide and receptor family, Pharmacol. Rev., 53 (2001) 381–415. Mollereau, C. and Mouledous, L., Tissue distribution of the opioid receptor-like (ORL1) receptor, Peptides, 21 (2000) 907–917. Nelson, R.M., Calo’, G., Guerrini, R., Hainsworth, A.H., Green, A.R. and Lambert, D.G., Nociceptin/orphanin FQ inhibits ischaemia-induced glutamate efflux from rat cerebrocortical slices, NeuroReport, 11 (2000) 3689–3692. Nicol, B., Lambert, D.G., Rowbotham, D.J., Smart, D. and McKnight, A.T., Nociceptin induced inhibition of K 1 evoked glutamate release from rat cerebrocortical slices, Br. J. Pharmacol, 119 (1996) 1081–1083. Nicol, B., Rowbotham, D.J. and Lambert, D.G., m and k opioids inhibit K 1 evoked glutamate release from rat cerebrocortical slices, Neurosci. Lett., 218 (1996) 79–82. Okawa, H., Hirst, R.A., Smart, D., McKnight, A.T. and Lambert, D.G., Rat central ORL-1 receptor uncouples from adenylyl cyclase during membrane preparation, Neurosci. Lett., 246 (1998) 49–52. 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 rat brain, rat vas deferens and CHO cells expressing recombinant human nociceptin receptors, Br. J. Pharmacol., 127 (1999) 123–130. Peluso, J., Laforge, K.S., Matthes, H.W., Kreek, M.J., Kieffer, B.L. and Gaveriaux-Ruff, C., Distribution of nociceptin/ orphanin FQ receptor transcript in human central nervous system and immune cells, J. Neuroimmunol., 81 (1998) 184–192. Reinscheid, R.K., Nothacker, H.-P., Bourson, A., Ardati, A., Henningsen, R.A., Bunzow, J.R., Grandy, D.K., Langen, H., Monsma Jr, F.J. and Civelli, O., Orphanin FQ: a neuropeptide that activates an opioid like G protein-coupled receptor, Science, 270 (1995) 792–794. Schlicker, E. and Morari, M., Nociceptin/orphanin FQ and neurotransmitter release in the central nervous system, Peptides, 21 (2000) 1023–1029. Talman, W.T., Dragon, D.N., Ohta, H. and Lin, L.H., Nitroxidergic influences on cardiovascular control by NTS: a link with glutamate, Ann. N. Y. Acad. Sci., 940 (2001) 169–178. Wang, J., Irnaten, M., Neff, R.A., Venkatesan, P., Evans, C., Loewy, A.D., Mettenleiter, T.C. and Mendelowitz, D., Synaptic and neurotransmitter activation of cardiac vagal neurones in the nucleus ambiguous, Ann. N. Y. Acad. Sci., 940 (2001) 237–246. Wang, J.B., Johnson, P.S., Imai, Y., Persico, A.M., Ozenberger, B.A., Eppler, C.M. and Uhl, G.R., cDNA cloning of an orphan opiate receptor gene family member and its splice variant, FEBS Lett., 348 (1994) 75–79. Wick, M.J., Minnerrath, S.R., Roy, S., Ramakrishnan, S. and Loh, H.H., Expression of alternate forms of brain orphan receptor mRNA in activated human peripheral blood lymphocytes and lymphocytic cell lines, Brain Res. Mol. Brain Res., 32 (1995) 342–347.
Nociceptin/orphanin FQ inhibits glutamate release from rat cerebellar and brain stem slices B. Nicol 1, D.J. Rowbotham, D.G. Lambert* University Department of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester LE1 5WW, UK Received 8 February 2002; received in revised form 12 March 2002; accepted 18 March 2002
Abstract We have previously demonstrated that nociceptin/orphanin FQ (N/OFQ), inhibits K 1 depolarisation-evoked glutamate release from rat cerebrocortical slices. In this study we have examined the effects of N/OFQ on glutamate release from rat cerebellar and brain stem slices as there are regional differences in nociceptin/orphanin FQ receptor (NOP) expression. Slices were depolarised with two pulses of 46 mM K 1 (S1 and S2) with N/OFQ added after S1. Glutamate (non-radioactive) was measured using a fluorescence-based assay. N/OFQ effects were assessed by measuring area under S1 and S2 release curves and calculation of S2/S1 ratios. In cerebellar slices K 1 evoked S2/S1 ratio was 1.17 ^ 0.10 (n ¼ 28). This was reduced in a concentration dependent (EC50 22 nM; Emax 46%) and naloxone (10 mM) insensitive manner by N/OFQ. In the brain stem K 1 evoked glutamate release was considerably reduced compared to cerebellum. In several preparations K 1 failed to evoke a significant release. In those that did K 1 evoked S2/S1 ratio was 1.03 ^ 0.07 (n ¼ 13). A total of 100 nM N/OFQ reduced this by 38 ^ 12% and this response was naloxone insensitive. Due to this small response and its variability we could not construct a full concentration response curve. In conclusion we have demonstrated a functional NOP in rat cerebellum and brain stem that inhibits the release of glutamate. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Glutamate release; Nociceptin/orphanin FQ; Cerebellar slice; Brain stem slice
Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the orphan opioid receptor-like receptor (NOP) [7,16]. This terminology is consistent with recent suggestions by IUPHAR [4]. N/OFQ is involved in many central (and peripheral) responses including pain processing, anxiolysis, feeding, motor and cardiovascular control [3,8]. NOP receptors are widely distributed in both tissues derived from the central nervous system; including cerebrocortex, cerebellum and brain stem [5,9] and a range of peripheral tissues including intestine, skeletal muscle, vas deferens, spleen and lymphocytes [1,15,20,21]. Indeed, in a series of radioligand binding studies to crude membranes prepared from rat cerebrocortes, cerebellum and brain stem using [ 125I]Y 14N/OFQ as a radioligand we reported expression of 180, 12 and 52 fmol/mg protein of NOP, respectively [13]. Very low levels of N/OFQ expression in the cerebellum are consistent with the literature [9] and it is well known * Corresponding author. Tel.: 144-116-258-5291; fax: 144-116285-4487. E-mail address: [email protected] (D.G. Lambert). 1 Present address: Animal Health Discovery, Pfizer, Sandwich, Kent, UK.
that this brain region is particularly rich in glutamatergic neurones, e.g. purkinje fibres and N/OFQ has effects on motor control (see refs [3,8]). In the brain stem there are several nuclei that express NOP including locus coeruleus, raphe magnus, nucleus ambiguous, dorsal motor nucleus of the vagus (DMNV) and nucleus of the tractus solitarii (NTS) (see ref. [9]). In both DMNV and NTS, involved in cardiovascular control, there is evidence for a role for glutamatergic transmission [18,19]. We have previously demonstrated that N/OFQ, N/ OFQ(1–13)NH2 and the partial agonist [F/G]N/OFQ(1– 13)NH2 inhibit the release of glutamate from rat cerebrocortical slices. The response to N/OFQ was insensitive to the opioid antagonist naloxone [11,14]. In addition we have also recently demonstrated that N/OFQ inhibits ischaemia induced glutamate release in the same preparation [10]. Moreover, N/OFQ has been shown to inhibit the release of a wide range of other neurotransmitters including noradrenaline, dopamine, serotonin and acetylcholine [17]. In this study we have examined the effects of N/OFQ on K 1-evoked release of endogenous glutamate from rat cerebellar and brain stem slices and compared these data to our
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 31 7- 8
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Fig. 1. Potassium (46 mM) evoked glutamate release from rat cerebellar slices is extracellular Ca 21 dependent and inhibited by nociceptin (N/OFQ). In panels A and B two 2 min pulses of 46 mM K 1 (S1 and S2) were applied to the slices resulting in monophasic releases of glutamate which are expressed relative to the mean of the first three fractions collected. In panel A immediately after S1 the buffer was replaced with nominally Ca 21 free solution containing 0.1 mM EGTA until the end of the experiment. In panel B immediately after S1 the buffer was replaced with solution containing 100 nM N/OFQ until the end of the experiment. In panel C, based on S2/S1 ratios, N/OFQ produced a concentration dependent inhibition of evoked release (whole curve P , 0:05) with an estimated EC50 of 22 nM. In panel D the response to 100 nM N/OFQ was insensitive to 10 mM naloxone (*P , 0:05 compared to control). Data are mean ^ SEM (n . 5).
previous study in the cerebrocortex as there are regional differences in NOP expression. Effects of N/OFQ on glutamate release from rat brain slices were examined essentially as described previously [11,14]. Briefly, the brain was removed from female Wistar rats (200–250 g) and the cerebellum and brain stem dissected. Typically tissue from two animals was pooled, this was particularly important for the brain stem due to its small size. Slices were cut separately and washed three times in 95% O2 and 5% CO2 Krebs buffer, pH 7.4, prior to agitation in a shaking water bath at 378C for 40 min. A total of 1 ml of gravity packed slices were pipetted into a perfusion chamber. Slices were perfused at 378C for 60 min at 1 ml/min prior to collection of 2 min fractions. A 2 min pulse of 46 mM K 1 (Na 1 adjusted) was applied (S1) following 6 min of perfusion. Slices were perfused for a further 30 min, prior to the second application of a 2 min pulse of 46
mM K 1 (S2). Perfusate glutamate concentrations were measured fluorimetrically as described previously [11,12,14] and expressed relative to the mean of the first three basal samples. Based on the raw data, area under the S1 and S2 release profiles and an S2/S1 ratio was calculated. Use of S2/S1 ratios allows for internal control of a variable S1 phase between tissues prepared from different animals and on different days. To examine the effects of N/OFQ on glutamate release, N/OFQ (in the presence of 30 mM amastatin, bestatin, captopril and phosphoramidon) was applied immediately after S1 until the end of the experiment (also present during S2). Agents inhibiting release will reduce S2 and hence the S2/S1 ratio. As it was not possible to construct a full concentration response curve in a single animal three conditions were tested in each experiment; control, high concentration of N/OFQ and low concentration of N/OFQ in random order. In some experiments, naloxone (10 mM)
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Fig. 2. Potassium (46 mM) evoked glutamate release from rat brain stem slices is inhibited by nociceptin (N/OFQ). In panel A two 2 min pulses of 46 mM K 1 (S1 and S2) were applied to the slices resulting in monophasic releases of glutamate which are expressed relative to the mean of the first three fractions collected. This was inhibited by 100 nM N/OFQ applied immediately after S1 until the end of the experiment. In panel B the response to 100 nM N/OFQ was insensitive to 10 mM naloxone (*P , 0:05 compared to control). Data are mean ^ SEM (n . 5).
and Ca 21 free buffer plus 0.1 mM EGTA were used to block classical (m, d and k) opioid receptors and confirm Ca 21 sensitive release respectively. N/OFQ was provided by Dr R. Guerrini of the University of Ferrara, Italy. Phosphoramidon was from peptide institute (Osaka, Japan). All other reagents were from Sigma Chemical Co. (Poole, UK). Data are presented as the mean ^ SEM (n, independent experiments). The pharmacological terminology adopted in this study is in line with the IUPHAR recommendations [6]: EC50 is concentration of an agonist that produces 50% of the maximum possible effect, Emax. All curve fitting was performed using GRAPHPAD-PRISM. Statistical comparisons of paired samples were made using Freidman’s analysis of variance followed by Dunn’s test or Wilcoxon Rank sum test as appropriate and considered significant when P , 0:05. A 2 min pulse of 46 mM K 1 produced a monophasic release of glutamate from rat cerebellar slices reaching some 2.5-fold over basal. A second pulse of K 1 also produced a similar release profile (Figs. 1A,B). In common with the cerebrocortex [12] K 1 evoked glutamate release was substantially extracellular Ca 21 dependent as removal of extracellular Ca 21 and addition of 0.1 mM EGTA reduced the S2/S1 ratio by some 70% compared to control (80% in cerebrocortex [12]) (Fig. 1A). In 28 control experiments in this tissue S2/S1 ratios were 1.17 ^ 0.10. When N/ OFQ was included prior to S2 the S2 release was reduced. An example at 100 nM N/OFQ is shown in Fig. 1B where a 40 ^ 9% (n ¼ 7) inhibition of release was observed. The effects of N/OFQ were concentration dependent with Emax and EC50 values of 46% and 22 nM, respectively (Fig. 1C). In the rat cerebrocortex we reported EC50 and Emax values for N/OFQ of 51 nM and 68%, respectively [11]. The N/ OFQ (100 nM) response was not due to activation of neuro-
nal opioid receptors as this was naloxone (10 mM) insensitive (Fig. 1D). In addition we were unable to detect any change in basal glutamate release immediately following N/OFQ application (data not shown). In the brain stem K 1 evoked glutamate release was considerably reduced compared to the cerebellum. In contrast to cerebellar slices in several preparations K 1 failed to evoke a significant release. In those (n ¼ 13) that did respond, we were able to detect monophasic release profiles in response to K 1 challenge during both S1 and S2 yielding an S2/S1 ratio of 1.03 ^ 0.07 (Fig. 2A). When N/OFQ was included prior to S2 the S2 release was reduced. An example at 100 nM N/OFQ is shown in Fig. 2A where a 38 ^ 12% (n ¼ 5) inhibition of release was observed, this was not significantly different (P . 0:05) from that observed at 100 nM in the cerebellum. In view of the small response and its variability we did not construct a full concentration response curve. At 100 nM N/OFQ, naloxone (10 mM) was ineffective (Fig. 2B). In this study we have clearly demonstrated that the peptide N/OFQ inhibits the release of glutamate from rat cerebellar and brain stem slices. This was mediated by the NOP as the response was insensitive to naloxone. Whilst it would have been preferable to examine the effects of the selective NOP antagonist [Nphe 1]N/OFQ(1–13)NH2 [2] this was not available to us at the time these studies were undertaken. In the cerebellum we were able to construct a full concentration response curve that yielded EC50 values similar to that previously reported in rat cerebrocortex [11]. However, in the brain stem such analysis was not possible. Reduced K 1-evoked glutamate release in the brain stem (and more specifically its absence in some preparations) compared to cerebellum or cerebrocortex [11] might suggest a high level of N/OFQ-ergic tone in this tissue
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such that endogenously released N/OFQ is sufficient to abolish evoked release. In view of the small degree of inhibition produced by addition of a relatively high concentration of exogenous N/OFQ this supposition is, in our view, unlikely. However, in the absence of studies utilising selective NOP antagonists (i.e. will pre-treatment with NOP antagonists increase the number of tissue preparations that respond and enhance the size of response in those that already do respond) this supposition cannot be completely ruled out. In the cortex we have previously demonstrated that N/ OFQ inhibits the release of glutamate in response to a depolarising challenge [11,14] in much the same way as m and k (but not d) opioids [12]. Coupled with the Ca 21 sensitivity, these data are in agreement with the results of the present study. In addition, we have also shown that N/OFQ inhibits ischaemia induced glutamate release and tentatively suggest that N/OFQ and the newer non-peptide agonists might possess neuroprotective properties. In conclusion we have demonstrated a functional NOP in rat cerebellum and brain stem that inhibits the release of glutamate. Further studies in the cerebellum with a range of non-peptide agonists and antagonists are warranted.
[8]
[9]
[10]
[11]
[12]
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