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Dynorphin displaces binding at the glycine site of the NMDA receptor in the rat striatum
Neuroscience Letters 415 (2007) 55–58
Dynorphin displaces binding at the glycine site of the NMDA receptor in the rat striatum Pieter Voorn a,∗ , Serge Valery van de Witte a , Ka wan Li b , Allert Jan Jonker a a
Department of Anatomy and Neurosciences, ICEN, Vrije Universiteit Medical Center, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands b Department of Molecular and Cellular Biology, Faculty of Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands Received 13 October 2006; received in revised form 18 December 2006; accepted 23 December 2006
Abstract Binding of dynorphin A (1–17 and 2–17) to NMDA receptors in the rat striatum was studied by displacing radioactive ligands for the receptor’s polyamine ([3 H]-Ifenprodil), glutamate ([3 H]-CGP-39653), dizocilpine ([3 H]-MK-801) and glycine ([3 H]-MDL105,519) sites with the neuropeptide. Dynorphin A selectively displaced [3 H]-MDL105,519 and none of the other ligands. Opioid antagonists did not affect displacement. Thus, in the striatum dynorphin may regulate NMDA receptor function via the glycineB site through non-opioid mechanisms. This may contribute to the long-term changes in behavioral responsiveness seen after dopamine depletion and treatment with dopaminomimetics which are associated with substantial changes in striatal dynorphin metabolism. © 2007 Published by Elsevier Ireland Ltd. Keywords: Basal ganglia; Glutamate receptor; Receptor binding; Opioid; GlycineB site
The administration of l-DOPA to rats with a unilaterally lesioned nigrostriatal dopaminergic system leads to an increased behavioral responsiveness to dopamine agonists [15]. In addition, this treatment induces an increase in the synthesis of the opioid peptide dynorphin in the dopamine-depleted striatum [27]. The rise in dynorphin mRNA levels is correlated with the intensity of the behavioral response, in the sense that higher levels of dynorphin are associated with a higher frequency of abnormal movements with dyskinetic characteristics [3]. The role of dynorphin in these behavioral processes is not clear. Infusion of low doses of dynorphin A (1–17) into the dopamine-depleted striatum prior to l-DOPA exposure increased sensitivity to a dopamine D1 receptor agonist, whereas high doses of the peptide strongly reduced D1 agonistinduced behavioral activity [26]. This suggests that dynorphin can interfere with (the development of) long-term changes in striatal dopaminergic neurotransmission. Comparable findings, with respect to a reduced behavioral response, come from
∗
Corresponding author. Tel.: +31 20 444 8051; fax: +31 20 444 8054. E-mail address: [email protected] (P. Voorn).
0304-3940/$ – see front matter © 2007 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2006.12.041
experiments on the effects of repeated administration of cocaine. Stimulation of the kappa opioid receptor, the opioid receptor for which dynorphin has highest affinity [18], blocked behavioral hyperresponsiveness and dopamine-induced immediate early gene expression that occurs after repeated cocaine [22,25]. These effects probably involve a kappa agonist-induced inhibition of dopamine release [6,24]. Same as in the cocaine studies mentioned above, kappa opioid receptor stimulation and altered dopamine release may be involved in the above-described effects of dynorphin in the dopamine-depleted and l-DOPA-treated animals. However, changes in dopaminergic neurotransmission may only partly explain the observed effects, since the kappa opioid receptor and dynorphin have been strongly implicated in glutamatergic neurotransmission [2,4,8,12,14,23,28]. NMDA receptor stimulation plays a crucial role in the sensitization of dopamine receptors, since MK-801, a non-competitive NMDA receptor antagonist, can block the l-DOPA-induced potentiation of turning behavior in unilaterally lesioned rats [16,27]. Interestingly, intrastriatal infusion of high doses of dynorphin, as pointed out above, had the same effect. Perhaps the modulatory (inhibitory and/or facilitatory) effects of dynorphin can be explained by a direct action of the peptide on the NMDA receptor.
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Therefore, we investigated in the present study if dynorphin binds to striatal NMDA receptors, which site of the NMDA receptor might be involved and whether a potential binding involves opioid or non-opioid mechanisms. Radioactive ligands were displaced with 10−9 to 10−3 M dynorphin A (1–17) and (2–17) (HPLC-purified; Bachem, King of Prussia, PA), with or without naloxone (Sigma–Aldrich, St. Louis, MO; 10−5 and 10−3 M) and nor-BNI (Sigma–Aldrich; 10−6 and 10−3 M) in cryostat sections (20 m) through the striatum from fresh-frozen rat brain tissue. Purity of dynorphin A samples was checked with electrospray (tandem) mass spectrometry which was performed on a Micromass Q-TOF mass spectrometer, as described previously [13]. The samples were dissolved in 0.1% formic acid/30% acetonitrile, and 10 l was loaded into a nanoelectrospray capillary, which was pulled from a borosilicate glass capillary GC 100F-10 with a micro-capillary puller. An internal wire electrode inserted inside the capillary was used for the measurement. Rats (male Wistar rats of ±250 g, Harlan, The Netherlands) were decapitated and the brains were dissected out on dry ice, in compliance with rulings of the Vrije Universiteit Animal Ethics Committee and EEC directive 86/609/EEC). Three rats were used for each experiment and per rat two sections were measured for each concentration of displacer ligand. Each experiment was repeated twice. Sections were thawed and incubated as follows. For [3 H]Ifenprodil, 15 min rinse at RT in 50 mM Tris–HCl, pH 7.4, incubation for 120 min in 20 nM [3 H]-Ifenprodil in 50 mM Tris–HCl with 3 M GBR 12909 and 3 M PPP, pH 7.4 at 4 ◦ C. Thereafter sections were washed 3 × 30 s in Tris–HCl at 4 ◦ C. For [3 H]-CGP-39653, sections were rinsed for 30 min at 0 ◦ C and preincubated for 20 min at 30 ◦ C in 50 mM Tris–acetate buffer, pH 7.6. Incubation was for 30 min in 20 nM [3 H]-CGP39653 in 50 mM Tris–acetate buffer with 1 mM MgCl2 , pH 7.6 at 0 ◦ C. After incubation sections were washed 20 s in the Tris–acetate buffer at 0 ◦ C. For [3 H]-MK-801, sections were rinsed for 30 min at 4 ◦ C in 50 mM Tris–HCl with 150 mM NaAc, pH 7.4. Incubation was for 120 min at RT in 5 nM [3 H]-MK-801 in 50 mM Tris–HCl with 150 mM NaAc, pH 7.4. Subsequently, sections were rinsed and then left in the Tris buffer for 80 min at 4 ◦ C. For [3 H]-MDL-105,519, sections were rinsed for 3 × 30 s at 0 ◦ C in 50 mM Tris–acetate, pH 7.4, incubated for 45 min at RT in 20 nM [3 H]-MDL-105,519 in Tris–acetate and rinsed for 3 × 30 s at 0 ◦ C in Tris–acetate, pH 7,4. The influence of the dissolvent DMSO on MDL-105,519 binding was corrected for at data points 10−3 M (1.12×, conc. DMSO 7.5%), 5 × 10−4 M (1.06×, conc. DMSO 3.76%), 10−4 M (0.94×, conc. DMSO 0.75%) and 5 × 10−5 M (0.94×, conc. DMSO 0.37%). After rinsing, sections were scraped off the slides for liquid scintillation counting (Wallac) during 10 min. GBR 12909 and PPP were from Sigma–Aldrich. All other chemicals were from Merck, Hawthorne, NY. Graphpad Prism 3 was used for all analyses and curve fitting for one site competition. Dynorphin (Dyn) A (1–17) purity was assessed with mass spectrometry and contamination with glycine could be excluded (Fig. 1). Dyn A (1–17) consistently gave the same results in the binding experiments as Dyn A (2–17). Therefore, only the results of Dyn A (1–17) will be shown.
Fig. 1. Mass spectrometric analyses of glycine and dynorphin samples demonstrate the absence of glycine in the dynorphin sample. (A) Dynorphin (1–17) sample contained low levels of several small molecules; molecular ion species corresponding to glycine was not observed. (B) Mass spectrum of sample containing glycine revealed the presence of a prominent molecular ion species (arrow) corresponding to glycine.
Dyn A did not displace the binding of [3 H]-MK-801 (Fig. 2). At low concentrations of dynorphin (10−8 to 10−6 M), there was a non-significant tendency for displacement. However, at higher concentrations binding of [3 H]-MK-801 was characterized by a potentiation at 10−4 M dynorphin. Neither [3 H]-Ifenprodil nor [3 H]-CGP-39653 could be displaced by dynorphin (Fig. 2). However, the peptide reliably displaced [3 H]-MDL-105,519, the ligand specific for the NMDA receptor’s glycine site (Fig. 3). KD for [3 H]-MDL-105,519 was established by displacing the ligand with its non-tritiated form; KD = 28 × 10−6 M. From this value, KI for dynorphin was calculated using the Cheng–Prussof formula; KI = 4 × 10−7 M. To determine if binding to the NMDA receptor’s glycine site is an opioid or non-opioid effect of dynorphin, incubations were carried out in the presence of low and high concentrations of the non-selective opioid receptor antagonist naloxone (10−5 M and 10−3 M) or the selective kappa opioid antagonist nor-BNI (10−6 M and 10−3 M). At 10−6 M, neither naloxone nor norBNI changed the displacement curve of [3 H]-MDL-105,519 by
Fig. 2. Dynorphin (1–17) does not affect binding of [3 H]-Ifenprodil and [3 H]CGP-39653. Binding of [3 H]-MK-801 was potentiated at 10−4 M dynorphin (1–17) (significantly different from all other data points in a post hoc Student Newman Keuls test – p < 0.05 – after oneway ANOVA). Data represent average ±S.E.M. percentage of total binding of three separate experiments. Data for [3 H]-MK-801 were fitted using spline fitting/LOWESS procedure.
P. Voorn et al. / Neuroscience Letters 415 (2007) 55–58
Fig. 3. Dynorphin (1–17) displaces [3 H]-MDL-105,519 (20 nM). IC50 = 5 × 10−7 M. Data represent average ±S.E.M. percentage of total binding of three separate experiments.
dynorphin (Fig. 4). In contrast, at high concentration (10−3 M) nor-BNI strongly potentiated the displacement of [3 H]-MDL105,519 (Fig. 4). The present study demonstrates in the striatum displacement of an NMDA antagonist selective for the glycineB site (MDL 105,519) by dynorphin. It could be ruled out, by using mass spectrometry analysis, that the observed displacement was caused by contamination of the dynorphin preparation with glycine. No effects were seen of dynorphin on the polyamine, dizocilpine or glutamate binding sites of the NMDA receptor, which may indicate that such interactions do not take place in the striatum as opposed to other regions [2,14,23]. The affinity of dynorphin for the glycineB site was found to be relatively low, which is related to the low affinity observed for MDL 105,519 in comparison to other studies [1,9,21]. A possible explanation may be that tissue sections were used instead of membrane preparations or cell culture. Evidence suggests that two glycineB sites exist, one with high (nanomolar) and one with low (micromolar) affinity [17]. Possibly, only the low affinity site was present in tissue sections.
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Both dynorphin A (1–17) and its non-opioid analogue (2–17) displaced MDL 105,519 binding. The effect could not be blocked by moderate concentrations of opioid antagonists, pointing to a non-opioid mechanism, as seen by others [2,12,14,23]. This finding is probably not the result of rapid breakdown of dynorphin A (1–17) to non-opioid derivatives since the peptide is apparently stable under the present experimental conditions [19]. However, effects were seen of high concentrations nor-BNI and naloxone and this suggests that all three classes of opioid receptors influence binding of dynorphin to the glycineB site. By binding to the glycineB site, dynorphin may regulate striatal NMDA receptor function in a similar way as in the hippocampus, where a glycine-dependent modulation of NMDA currents by dynorphin was observed [10,29]. Under physiological conditions the glycineB site is probably not saturated [5], so dynorphin may compete with glycine for binding to the NMDA receptor. The substantial changes in dynorphin metabolism that are induced by a 6-OHDA lesion and subsequent treatment with dopaminomimetics might strongly affect striatal NMDA receptor function. After the lesion, dynorphin levels in the striatum have decreased considerably [7,27]. A low dosage of dynorphin infused into the striatum at this time-point resulted in a potentiated response to a dopamine D1 receptor agonist after l-DOPA treatment [26]. The present results suggest that, in line with the findings of Zhang et al. [29], Jarvis et al. [10] and Lai et al. [12], this effect might be based on an enhancement of NMDA receptor function. In contrast, the reduced behavioral response after a high dosage of dynorphin [26] is difficult to explain on basis of the literature. Jarvis et al. [10] reported a depression of NMDA currents by dynorphin at high concentrations of glycine, but it is not known if the observed effects of dynorphin are concentration dependent. An inhibitory effect of dynorphin in high concentrations on NMDA receptor function can neither be excluded nor included. It must be noted that most reports concern an inhibitory effect of dynorphin on NMDA receptors and glutamate neurotransmission, albeit via mechanisms different from the non-opioid interactions reported in the present paper (e.g. [4,8,11,20,28]). In conclusion, our findings demonstrate that in the striatum dynorphin A may regulate NMDA receptor function through a non-opioid interaction at the glycineB binding site. This regulatory mechanism may be of specific importance when dynorphin levels strongly fluctuate as a consequence of changes in dopaminergic neurotransmission.
References
Fig. 4. Displacement of [3 H]-MDL-105,519 (20 nM) by dynorphin (1–17) in the presence of 10−5 and 10−3 M naloxone or 10−6 and 10−3 M nor-BNI. IC50 of dynorphin in the presence of 10−6 M nor-BNI is 1.2 × 10−7 M whereas IC50 in the presence of 10−3 M nor-BNI is 1.1 × 10−9 M. Data represent average ±S.E.M. percentage of total binding in three separate experiments.
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Dynorphin displaces binding at the glycine site of the NMDA receptor in the rat striatum Pieter Voorn a,∗ , Serge Valery van de Witte a , Ka wan Li b , Allert Jan Jonker a a
Department of Anatomy and Neurosciences, ICEN, Vrije Universiteit Medical Center, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands b Department of Molecular and Cellular Biology, Faculty of Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands Received 13 October 2006; received in revised form 18 December 2006; accepted 23 December 2006
Abstract Binding of dynorphin A (1–17 and 2–17) to NMDA receptors in the rat striatum was studied by displacing radioactive ligands for the receptor’s polyamine ([3 H]-Ifenprodil), glutamate ([3 H]-CGP-39653), dizocilpine ([3 H]-MK-801) and glycine ([3 H]-MDL105,519) sites with the neuropeptide. Dynorphin A selectively displaced [3 H]-MDL105,519 and none of the other ligands. Opioid antagonists did not affect displacement. Thus, in the striatum dynorphin may regulate NMDA receptor function via the glycineB site through non-opioid mechanisms. This may contribute to the long-term changes in behavioral responsiveness seen after dopamine depletion and treatment with dopaminomimetics which are associated with substantial changes in striatal dynorphin metabolism. © 2007 Published by Elsevier Ireland Ltd. Keywords: Basal ganglia; Glutamate receptor; Receptor binding; Opioid; GlycineB site
The administration of l-DOPA to rats with a unilaterally lesioned nigrostriatal dopaminergic system leads to an increased behavioral responsiveness to dopamine agonists [15]. In addition, this treatment induces an increase in the synthesis of the opioid peptide dynorphin in the dopamine-depleted striatum [27]. The rise in dynorphin mRNA levels is correlated with the intensity of the behavioral response, in the sense that higher levels of dynorphin are associated with a higher frequency of abnormal movements with dyskinetic characteristics [3]. The role of dynorphin in these behavioral processes is not clear. Infusion of low doses of dynorphin A (1–17) into the dopamine-depleted striatum prior to l-DOPA exposure increased sensitivity to a dopamine D1 receptor agonist, whereas high doses of the peptide strongly reduced D1 agonistinduced behavioral activity [26]. This suggests that dynorphin can interfere with (the development of) long-term changes in striatal dopaminergic neurotransmission. Comparable findings, with respect to a reduced behavioral response, come from
∗
Corresponding author. Tel.: +31 20 444 8051; fax: +31 20 444 8054. E-mail address: [email protected] (P. Voorn).
0304-3940/$ – see front matter © 2007 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2006.12.041
experiments on the effects of repeated administration of cocaine. Stimulation of the kappa opioid receptor, the opioid receptor for which dynorphin has highest affinity [18], blocked behavioral hyperresponsiveness and dopamine-induced immediate early gene expression that occurs after repeated cocaine [22,25]. These effects probably involve a kappa agonist-induced inhibition of dopamine release [6,24]. Same as in the cocaine studies mentioned above, kappa opioid receptor stimulation and altered dopamine release may be involved in the above-described effects of dynorphin in the dopamine-depleted and l-DOPA-treated animals. However, changes in dopaminergic neurotransmission may only partly explain the observed effects, since the kappa opioid receptor and dynorphin have been strongly implicated in glutamatergic neurotransmission [2,4,8,12,14,23,28]. NMDA receptor stimulation plays a crucial role in the sensitization of dopamine receptors, since MK-801, a non-competitive NMDA receptor antagonist, can block the l-DOPA-induced potentiation of turning behavior in unilaterally lesioned rats [16,27]. Interestingly, intrastriatal infusion of high doses of dynorphin, as pointed out above, had the same effect. Perhaps the modulatory (inhibitory and/or facilitatory) effects of dynorphin can be explained by a direct action of the peptide on the NMDA receptor.
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Therefore, we investigated in the present study if dynorphin binds to striatal NMDA receptors, which site of the NMDA receptor might be involved and whether a potential binding involves opioid or non-opioid mechanisms. Radioactive ligands were displaced with 10−9 to 10−3 M dynorphin A (1–17) and (2–17) (HPLC-purified; Bachem, King of Prussia, PA), with or without naloxone (Sigma–Aldrich, St. Louis, MO; 10−5 and 10−3 M) and nor-BNI (Sigma–Aldrich; 10−6 and 10−3 M) in cryostat sections (20 m) through the striatum from fresh-frozen rat brain tissue. Purity of dynorphin A samples was checked with electrospray (tandem) mass spectrometry which was performed on a Micromass Q-TOF mass spectrometer, as described previously [13]. The samples were dissolved in 0.1% formic acid/30% acetonitrile, and 10 l was loaded into a nanoelectrospray capillary, which was pulled from a borosilicate glass capillary GC 100F-10 with a micro-capillary puller. An internal wire electrode inserted inside the capillary was used for the measurement. Rats (male Wistar rats of ±250 g, Harlan, The Netherlands) were decapitated and the brains were dissected out on dry ice, in compliance with rulings of the Vrije Universiteit Animal Ethics Committee and EEC directive 86/609/EEC). Three rats were used for each experiment and per rat two sections were measured for each concentration of displacer ligand. Each experiment was repeated twice. Sections were thawed and incubated as follows. For [3 H]Ifenprodil, 15 min rinse at RT in 50 mM Tris–HCl, pH 7.4, incubation for 120 min in 20 nM [3 H]-Ifenprodil in 50 mM Tris–HCl with 3 M GBR 12909 and 3 M PPP, pH 7.4 at 4 ◦ C. Thereafter sections were washed 3 × 30 s in Tris–HCl at 4 ◦ C. For [3 H]-CGP-39653, sections were rinsed for 30 min at 0 ◦ C and preincubated for 20 min at 30 ◦ C in 50 mM Tris–acetate buffer, pH 7.6. Incubation was for 30 min in 20 nM [3 H]-CGP39653 in 50 mM Tris–acetate buffer with 1 mM MgCl2 , pH 7.6 at 0 ◦ C. After incubation sections were washed 20 s in the Tris–acetate buffer at 0 ◦ C. For [3 H]-MK-801, sections were rinsed for 30 min at 4 ◦ C in 50 mM Tris–HCl with 150 mM NaAc, pH 7.4. Incubation was for 120 min at RT in 5 nM [3 H]-MK-801 in 50 mM Tris–HCl with 150 mM NaAc, pH 7.4. Subsequently, sections were rinsed and then left in the Tris buffer for 80 min at 4 ◦ C. For [3 H]-MDL-105,519, sections were rinsed for 3 × 30 s at 0 ◦ C in 50 mM Tris–acetate, pH 7.4, incubated for 45 min at RT in 20 nM [3 H]-MDL-105,519 in Tris–acetate and rinsed for 3 × 30 s at 0 ◦ C in Tris–acetate, pH 7,4. The influence of the dissolvent DMSO on MDL-105,519 binding was corrected for at data points 10−3 M (1.12×, conc. DMSO 7.5%), 5 × 10−4 M (1.06×, conc. DMSO 3.76%), 10−4 M (0.94×, conc. DMSO 0.75%) and 5 × 10−5 M (0.94×, conc. DMSO 0.37%). After rinsing, sections were scraped off the slides for liquid scintillation counting (Wallac) during 10 min. GBR 12909 and PPP were from Sigma–Aldrich. All other chemicals were from Merck, Hawthorne, NY. Graphpad Prism 3 was used for all analyses and curve fitting for one site competition. Dynorphin (Dyn) A (1–17) purity was assessed with mass spectrometry and contamination with glycine could be excluded (Fig. 1). Dyn A (1–17) consistently gave the same results in the binding experiments as Dyn A (2–17). Therefore, only the results of Dyn A (1–17) will be shown.
Fig. 1. Mass spectrometric analyses of glycine and dynorphin samples demonstrate the absence of glycine in the dynorphin sample. (A) Dynorphin (1–17) sample contained low levels of several small molecules; molecular ion species corresponding to glycine was not observed. (B) Mass spectrum of sample containing glycine revealed the presence of a prominent molecular ion species (arrow) corresponding to glycine.
Dyn A did not displace the binding of [3 H]-MK-801 (Fig. 2). At low concentrations of dynorphin (10−8 to 10−6 M), there was a non-significant tendency for displacement. However, at higher concentrations binding of [3 H]-MK-801 was characterized by a potentiation at 10−4 M dynorphin. Neither [3 H]-Ifenprodil nor [3 H]-CGP-39653 could be displaced by dynorphin (Fig. 2). However, the peptide reliably displaced [3 H]-MDL-105,519, the ligand specific for the NMDA receptor’s glycine site (Fig. 3). KD for [3 H]-MDL-105,519 was established by displacing the ligand with its non-tritiated form; KD = 28 × 10−6 M. From this value, KI for dynorphin was calculated using the Cheng–Prussof formula; KI = 4 × 10−7 M. To determine if binding to the NMDA receptor’s glycine site is an opioid or non-opioid effect of dynorphin, incubations were carried out in the presence of low and high concentrations of the non-selective opioid receptor antagonist naloxone (10−5 M and 10−3 M) or the selective kappa opioid antagonist nor-BNI (10−6 M and 10−3 M). At 10−6 M, neither naloxone nor norBNI changed the displacement curve of [3 H]-MDL-105,519 by
Fig. 2. Dynorphin (1–17) does not affect binding of [3 H]-Ifenprodil and [3 H]CGP-39653. Binding of [3 H]-MK-801 was potentiated at 10−4 M dynorphin (1–17) (significantly different from all other data points in a post hoc Student Newman Keuls test – p < 0.05 – after oneway ANOVA). Data represent average ±S.E.M. percentage of total binding of three separate experiments. Data for [3 H]-MK-801 were fitted using spline fitting/LOWESS procedure.
P. Voorn et al. / Neuroscience Letters 415 (2007) 55–58
Fig. 3. Dynorphin (1–17) displaces [3 H]-MDL-105,519 (20 nM). IC50 = 5 × 10−7 M. Data represent average ±S.E.M. percentage of total binding of three separate experiments.
dynorphin (Fig. 4). In contrast, at high concentration (10−3 M) nor-BNI strongly potentiated the displacement of [3 H]-MDL105,519 (Fig. 4). The present study demonstrates in the striatum displacement of an NMDA antagonist selective for the glycineB site (MDL 105,519) by dynorphin. It could be ruled out, by using mass spectrometry analysis, that the observed displacement was caused by contamination of the dynorphin preparation with glycine. No effects were seen of dynorphin on the polyamine, dizocilpine or glutamate binding sites of the NMDA receptor, which may indicate that such interactions do not take place in the striatum as opposed to other regions [2,14,23]. The affinity of dynorphin for the glycineB site was found to be relatively low, which is related to the low affinity observed for MDL 105,519 in comparison to other studies [1,9,21]. A possible explanation may be that tissue sections were used instead of membrane preparations or cell culture. Evidence suggests that two glycineB sites exist, one with high (nanomolar) and one with low (micromolar) affinity [17]. Possibly, only the low affinity site was present in tissue sections.
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Both dynorphin A (1–17) and its non-opioid analogue (2–17) displaced MDL 105,519 binding. The effect could not be blocked by moderate concentrations of opioid antagonists, pointing to a non-opioid mechanism, as seen by others [2,12,14,23]. This finding is probably not the result of rapid breakdown of dynorphin A (1–17) to non-opioid derivatives since the peptide is apparently stable under the present experimental conditions [19]. However, effects were seen of high concentrations nor-BNI and naloxone and this suggests that all three classes of opioid receptors influence binding of dynorphin to the glycineB site. By binding to the glycineB site, dynorphin may regulate striatal NMDA receptor function in a similar way as in the hippocampus, where a glycine-dependent modulation of NMDA currents by dynorphin was observed [10,29]. Under physiological conditions the glycineB site is probably not saturated [5], so dynorphin may compete with glycine for binding to the NMDA receptor. The substantial changes in dynorphin metabolism that are induced by a 6-OHDA lesion and subsequent treatment with dopaminomimetics might strongly affect striatal NMDA receptor function. After the lesion, dynorphin levels in the striatum have decreased considerably [7,27]. A low dosage of dynorphin infused into the striatum at this time-point resulted in a potentiated response to a dopamine D1 receptor agonist after l-DOPA treatment [26]. The present results suggest that, in line with the findings of Zhang et al. [29], Jarvis et al. [10] and Lai et al. [12], this effect might be based on an enhancement of NMDA receptor function. In contrast, the reduced behavioral response after a high dosage of dynorphin [26] is difficult to explain on basis of the literature. Jarvis et al. [10] reported a depression of NMDA currents by dynorphin at high concentrations of glycine, but it is not known if the observed effects of dynorphin are concentration dependent. An inhibitory effect of dynorphin in high concentrations on NMDA receptor function can neither be excluded nor included. It must be noted that most reports concern an inhibitory effect of dynorphin on NMDA receptors and glutamate neurotransmission, albeit via mechanisms different from the non-opioid interactions reported in the present paper (e.g. [4,8,11,20,28]). In conclusion, our findings demonstrate that in the striatum dynorphin A may regulate NMDA receptor function through a non-opioid interaction at the glycineB binding site. This regulatory mechanism may be of specific importance when dynorphin levels strongly fluctuate as a consequence of changes in dopaminergic neurotransmission.
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Fig. 4. Displacement of [3 H]-MDL-105,519 (20 nM) by dynorphin (1–17) in the presence of 10−5 and 10−3 M naloxone or 10−6 and 10−3 M nor-BNI. IC50 of dynorphin in the presence of 10−6 M nor-BNI is 1.2 × 10−7 M whereas IC50 in the presence of 10−3 M nor-BNI is 1.1 × 10−9 M. Data represent average ±S.E.M. percentage of total binding in three separate experiments.
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