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Affinity Labeling of δ Opioid Receptors by an Enkephalin-Derivative Alkylating Agent, DSLET-Mal
Biochemical and Biophysical Research Communications 265, 513–519 (1999) Article ID bbrc.1999.1682, available online at http://www.idealibrary.com on
Affinity Labeling of d Opioid Receptors by an EnkephalinDerivative Alkylating Agent, DSLET-Mal Ildiko´ Szatma´ri,* Gyo¨rgy Orosz,† Ka´lma´n Medzihradszky,† and Anna Borsodi* ,1 *Institute of Biochemistry, Biological Research Center and †Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Szeged, Hungary
Received October 4, 1999
Opioid binding properties of Tyr-D-Ser-Gly-Phe-LeuThr-NH-NH-Gly-Mal (DSLET-Mal), a novel enkephalinframed affinity label, was determined in rat brain membranes. In competition studies the ligand showed high affinity for the delta opioid sites, labelled by [ 3H][Ile 5,6]deltorphin II (K i 5 8 nM), whereas its binding to the mu ([ 3H]DAMGO) and kappa ([ 3H]EKC) sites was weaker. Preincubation of the rat brain membranes with DSLET-Mal at micromolar concentrations resulted in a wash-resistant and dose-dependent inhibition of the [ 3H][Ile 5,6]deltorphin II binding sites (96% blocking at 10 mM concentration). Intracerebroventricular (ICV) administration of DSLET-Mal reduced the density of delta opioid receptors and had no effect on mu and kappa receptors, as determined by saturation binding studies. [Ile 5,6]deltorphin II-stimulated [ 35S]GTPgS binding was determined in membrane preparations of different brain areas of the ICV-treated animals. In both frontal cortex and hippocampus DSLET-Mal significantly decreased G protein activation by the delta agonist, having no effect on DAMGO stimulated [ 35S]GTPgS binding. DSLET-Mal had qualitatively similar effects on both receptor binding and G protein activation. These characteristics of the compound studied suggest that DSLETMal can serve as an affinity label for further studies of the delta-opioid receptors. © 1999 Academic Press
Opioid receptors belong to the superfamily of G protein coupled receptors. They are integral membrane proteins characterised by amino acid sequences, which contain seven hydrophobic domains, representing the transmembrane regions. Opioid agonists stimulate the exchange of guanosine triphosphate (GTP) for guanosine diphosphate (GDP) on binding to their re1 To whom correspondence should be addressed at Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary. Fax: 36-62-433432. E-mail: [email protected].
ceptor by increasing the affinity of the Ga subunit of the G protein for GTP versus GDP. The GTP-Ga complex can than interact with intracellular effectors until an intrinsic GTPase on the Ga subunit hydrolyses GTP to GDP and the cycle repeats (1). The peptides Leu-enkephalin and Met-enkephalin are the naturally occurring ligands for the delta-opioid receptor (2). In efforts to produce more stable and selective ligands, several analogs of enkephalin have been synthesised such as: [D-Ala 2, D-Leu 5]enkephalin (DADLE) (3), [D-Thr 2, Leu 5, Thr 6]enkephalin (DTLET) (4) and [D-Ser 2, Leu 5, Thr 6]enkephalin (DSLET) (5). These peptides contain D-amino acids, the incorporation of which has been shown to stabilise peptides against proteolytic inactivation (6, 7). Opioid receptors have been shown to contain a thiol group at, or close to the ligand binding site (8, 9, 10). Alkylation of these sites by N-ethylmaleimid inhibits binding of mu and delta ligands, and the site is protected from alkylation by the bound ligand. The receptor thiol group is likely to be the nucleophile, which attacks receptor-specific, electrophilic affinity labels. Affinity ligands that form covalent bonds with receptors have been very useful in the elucidation of receptor structure (11). Many attempts have been made to prepare site directed irreversible probes for opioid receptors using alkaloid and peptide ligands. Alteration of free carboxyl termini of opioid peptides with functional groups resulted in ligands capable of interacting with the receptor in a wash-resistant manner. Peptides were modified with a number of reacting groups, including chloromethylketone (12, 13), melphalan (14, 15) and azido groups (16, 17). As thiol groups are known to easily react by addition to the maleoyl functional group, maleoyl group containing peptides were designed and tested in receptor binding studies. The maleoyl function is a highly reactive group, structurally it is very similar to the fumarate methyl ester, which was utilised in b-funaltrexamine (18), a widely utilised mu opioid irreversible ligand.
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The different maleoyl enkephalin analogues presented good selectivity to the delta opioid receptor and blocked a high percentage of these binding sites (19). In this study we determined the binding properties of the maleoylated DSLET in ligand binding and [ 35S]GTPgS binding assays. Furthermore, we investigated the effects of intracerebroventricular administration of this compound on agonist-stimulated [ 35S]GTPgS binding in rat brain membranes. As both frontal cortex and hippocampus are known to be enriched in delta opioid receptors (20), these two brain regions were selected for the functional assays. MATERIALS AND METHODS Chemicals. [ 3H]Ethylketocyclazocine (EKC; 20 Ci/mmol) was purchased from DuPont NEN (Cambridge, MA). [3H][D-Ala2(Me)Phe4Gly5ol]enkephalin (DAMGO; 55 Ci/mmol) was from Amersham, UK. [ 3H]naloxone (35 Ci/mmol) and [ 3H][Ile 5,6]deltorphin II (49 Ci/mmol) were synthesised in the Isotope Laboratory of Biological Research Center, Szeged (21, 22). All other chemicals used in this study were of analytical grade and purchased from Sigma (St. Louis, MO) or Reanal/Egis Pharmaceuticals (Budapest, Hungary). Synthesis of DSLET-Mal. Boc-Tyr(tBu)-D-Ser(tBu)-Gly-Phe-LeuThr(tBu)-OH was synthesized on 2-chlorotritylchloride resin (23) by Fmoc-technique. The details of the peptide synthesis is given elsewhere (24). The protected peptide acid was converted to its methyl ester by diazomethane and then reacted with hydrazine hydrate in methanol to obtain Boc-Tyr(tBu)-D-Ser(tBu)-Gly-Phe-Leu-Thr(tBu)NH-NH 2. This compound was reacted with maleoyl glycine in the presence of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate and diisopropyl-ethylamine in N,N-dimethyl formamide. The protecting groups were removed in trifluoroacetic acid containing 10% anisole. The crude product purified by preparative high performance liquid chromatography and analyzed by amino acid analysis and FAB-mass spectrometry. Intracerebroventricular (ICV) administration of peptides. Male Wistar rats (250 –300 g) were implanted with 20 G stainless steel canulae into the 3rd ventricle (1.3 mm rostral and 1.5 mm lateral from bregma, 3.5 mm ventral from the skull surface). Following 3– 4 days of recovery from surgery, animals were administered either 0 or 40 nmol of DSLET or DSLET-Mal in 4 ml of saline. Animals were sacrificed 24 h after the ICV administration, brain sections were removed and processed. Preparation of rat brain membranes. A crude membrane fraction was prepared from Wistar rat brains according to an earlier published method (25). The protein concentration was determined by the Bradford method (Bradford, 1986) (26), using bovine serum albumin as standard. Pretreatment of membranes (affinity labelling). Freshly prepared rat brain membranes (0.4 – 0.8 mg/ml) in 50 mM Tris-HCl (pH 7.4) buffer were preincubated with the compound to be tested at the concentrations indicated at 0°C for 1 h, in a final volume of 1 ml. Control tubes were run without the studied compound. After preincubation the mixtures were diluted to 1:15 with the same buffer and centrifuged (25000 g, 10 min). The pellets were resuspended in the buffer and incubated again for 15 min at 0°C. This washing step (centrifugation and resuspension) was repeated 4 times. The final pellets were resuspended in 4 ml of buffer and used immediately for [ 3H]naloxone binding assay in order to determine the amount of the irreversibly labelled binding sites. To determine the efficiency of the washing procedure, membranes were also preincubated with naloxone (reversible opioid ligand).
TABLE 1
Binding Affinities of DSLET and DSLET-Mal at Three Representative Opioid Ligand Binding Sites in Rat Brain Membranes K i (nM) Ligand
[ 3H]DAMGO
[ 3H][Ile 5,6]deltorphin II
[ 3H]EKC
DSLET DSLET-Mal
250 6 17 94 6 12
6.3 6 1.6 8.0 6 1.5
n.d. 147 6 24
Note. Rat brain membranes were incubated with the radioligands in the presence of the unlabelled peptide in 10 concentrations, ranging from 0.01 to 10000 nM. Assays were carried out as described under Materials and Methods. Values are averages of at least three experiments carried out in duplicate. n.d., not determined.
Receptor binding assay. The experiments were performed in glass for [ 3H]naloxone, [ 3H]EKC and [ 3H]DAMGO or plastic tubes for [ 3H][Ile 5,6]deltorphin II in 50 mM Tris-HCl buffer (pH 7.4) in a final volume of 1 ml and at a protein concentration of 0.3– 0.5 mg/ml. In case of [ 3H]EKC, the binding was performed in presence of 100 nM DAMGO and 100 nM [D-Ala 2, Leu 5]enkephalin in order to block the crossbinding to mu and delta sites. Incubation was started by addition of membrane suspension and terminated by rapid vacuum filtration through Whatman GF/C (for [ 3H][Ile 5,6]deltorphin II and [ 3H]DAMGO) or GF/B (in case of [ 3H]naloxone and [ 3H]EKC) filters using a Brandel Cell Harvester. After three washes with 5 ml portions of ice-cold buffer, the filters were dried at 37°C. The radioactivity was measured in a toluene based scintillation cocktail using a Wallac 1409 scintillation counter. The displacement curves were analyzed with the GraFit 3.0 using a nonlinear least squares algorithm for one site model. All experiments were carried out as duplicate assays and repeated at least three times. The given values are means 6 SEM. [ 35S]GTPgS binding. GTPgS is a thiol derivative of GTP that is resistant to hydrolysis by the GTPase activity of Ga. Opioid stimulation of [ 35S]GTPgS binding is an effective assay for G protein activation by opioids in vitro, using brain homogenates. Tubes containing 10 mg of protein, 30 mM GDP, 10 29–10 25 M opioid ligands, and 0.05 nM [ 35S]GTPgS, all in 50 mM Tris-HCl buffer containing 1 mM EGTA, 100 mM NaCl and 3 mM MgCl 2 in a final volume of 1 ml were incubated for 1 h, at 30°C. Non-stimulated activity was measured in the absence of tested compounds, non-specific binding was measured in the presence of 100 mM unlabelled GTPgS. The incubation was started by the addition of [ 35S]GTPgS and was terminated by filtrating the samples through Whatman GF/F glass fibre filters. Filters were washed three times with 5 ml of ice-cold 50 mM TrisHCl buffer (pH 7.4) in a Millipore filtration instrument, then dried. Bound radioactivity was measured in a Wallac 1409 scintillation counter using a toluene based scintillation cocktail. Stimulation is given as percent of the specific binding. Data were calculated from three independent experiments performed in triplicates.
RESULTS The ability of DSLET-Mal to compete reversibly with the binding of type specific opioid radioligands was determined over a wide concentration range of the studied ligand. The equilibrium inhibition constants (K i values) are shown in Table 1. DSLET-Mal bound with the highest affinity (K i 5 8.0 nM) to the
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FIG. 1. Inhibition of [ 3H][Ile 5,6]deltorphin II binding to rat brain membrane by various delta selective peptide ligands: (■) [Ile 5,6]deltorphin II, (Œ) [D-Ala 2, Leu 5]enkephalin, () DSLET, (F) DSLETMal, (p) DAMGO.
[ 3H][Ile 5,6]deltorphin II (delta) sites and had much lower potency in inhibiting both [ 3H]DAMGO (mu) and [ 3H]EKC (kappa) binding. Compared to the parent compound, the maleoylated peptide has the mu/delta selectivity ratio in the same range. Figure 1 shows the potency in inhibiting the [ 3H][Ile 5,6]deltorphin II binding of DSLET-Mal compared to other delta specific peptide ligands. As it is its affinity at the delta site was comparable to that of the other analogues. Sodium ions at 100 mM concentration decreased the affinity of DSLET-Mal suggesting its agonist character (28). The [ 35S]GTPgS binding assays showed its agonist character too: DSLET-Mal stimulated the GTPgS binding and the fact that addition of DSLET-Mal to [Ile 5,6]deltorphin II stimulated G protein activation had no effect on the reaction, excluded the antagonist property of the ligand. In order to test the irreversible binding of the studied ligand, rat brain membranes were prelabelled with
DSLET-Mal, then extensively washed to remove the reversibly bound ligand. Ligands producing a high degree of receptor occupation after this dissociation step are likely to be covalently attached. The irreversible binding of DSLET-Mal to rat brain membrane was tested with [ 3H][Ile 5,6]deltorphin II and compared to the effect of its parent compound DSLET. As shown in Fig. 2A, DSLET-Mal caused a dose-dependent, wash-resistant inhibition of the specific binding of [ 3H][Ile 5,6]deltorphin II. At 10 mM concentration a 96% irreversible blockade was observed. In the case of DSLET no such effect was observed. The receptortype selectivity of the irreversible binding was estimated using mu ([ 3H]DAMGO), delta ([ 3H][Ile 5,6]deltorphin II) and kappa ([ 3H]EKC) specific radioligands. Figure 2B shows the receptor types occupied in DSLET-Mal pretreated membranes. The effect of DSLET-Mal on receptor loss was moderately selective for delta receptors. Equilibrium saturation binding studies with [3H][Ile5,6]deltorphin II were also carried out with DSLET-Mal treated and control membranes (Fig. 3). Specific binding of [ 3H][Ile 5,6]deltorphin II was characterised by a single high affinity site. The maximal number of binding sites (B max) was substantially decreased in membranes preincubated with 10 mM affinity reagent. While in the case of the control membranes the B max showed to be 102.5 fmol/mg protein, on the pretreated membranes the value was just 19.4 fmol/mg. The equilibrium dissociation constant (K d) value of [ 3H][Ile 5,6]deltorphin II was not affected significantly by the presence of the maleoylated peptide, both in control and pretreated membranes K d presented values in subnanomolar range, 0.63 and 0.74 nM, respectively.
FIG. 2. Irreversible interaction of DSLET-Mal with different opioid receptor types. Rat brain membranes were preincubated with the respective peptide and then treated as described under Materials and Methods. Following this pretreatment the recovery of receptors was assessed using [ 3H]-ligands. Values are expressed in comparison to controls, which were preincubated without the peptide, but otherwise treated the same way. (A) The concentration dependence of irreversible interaction of the parent and maleoylated peptide with the delta opioid sites. (B) The receptor selectivity of the irreversible effect. Mu receptors were labelled with [ 3H]DAMGO (Œ), delta receptors were labelled with [ 3H][Ile 5,6]deltorphin II (■), and kappa receptors were labelled with [ 3H]EKC ().
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FIG. 3. Saturation binding isotherm (A) and Scatchard plot (B) of [ 3H][Ile 5,6]deltorphin II specific binding in control (■) and DSLET-Mal treated (h) membranes. In control experiments the membrane was preincubated without the peptide, but otherwise treated the same way.
In the next set of experiments we studied the effect of intracerebroventricular administration of DSLET-Mal on mu- and delta-agonist stimulated [ 35S]GTPgS binding. Firstly, equilibrium saturation binding studies with both [ 3H][Ile 5,6]deltorphin II, [ 3H]DAMGO and [ 3H]EKC were carried out in order to see the effect of DSLET-Mal on the density of the opioid receptor types. The intracerebroventricular administration of DSLETMal resulted in a decrease in the maximal number of binding sites available to the delta-specific radioligand, without significantly changing its affinity (K d). In order to see the significance of the decrease in the B max value, unpaired t-tests were performed. The obtained twotailed p value showed to be 0.0078. As shown in Table 2, the in vivo pretreatment did not significantly alter neither the affinity, nor the B max of the mu and kappa radioligands. The same experiments were carried out using the parent compound. The intracerebroventricular administration of DSLET had no effect neither on the maximal number of binding sites, nor on the equilibrium binding constant of the [ 3H][Ile 5,6]deltorphin II binding (Table 2). The mu- and delta-agonist stimulated [ 35S]GTPgS binding was carried out on the frontal cortici and hippocampi of control and ICV-treated animals. In both brain regions DSLET-Mal administration significantly decreased G protein activation by the delta agonist [Ile 5,6]deltorphin II, having no effect on DAMGOstimulated [ 35S]GTPgS binding (Fig. 4, Table 3). To see the irreversibility of the ICV administered compound, the membranes were washed as in the in vitro labelling experiments, and the same [ 35S]GTPgS binding experiments were carried out. The effect of DSLET-Mal remained after several washings. When the parent compound, DSLET was administered, no differences could be observed between the control and treated mem-
branes, neither in mu-, nor in delta-agonist stimulated [ 35S]GTPgS binding (data not shown). DISCUSSION A novel type of enkephalin-framed affinity label was prepared and tested for its activity both in vitro and in vivo. The potency of the binding of DSLET-Mal to the opioid receptors was investigated in competition experiments using crude membrane fraction of rat brain. The presence of the maleoyl group at the C terminus of the peptide did not change significantly the binding
TABLE 2
The Effect of Intracerebroventricular Administration of DSLET-Mal and DSLET on the Binding Constants (K d) and Maximal Binding Sites (B max) of Type-Specific Opioid Radioligands Ligand [ 3H][Ile 5,6]deltorphin II Control 1DSLET-Mal 1DSLET [ 3H]DAMGO Control 1DSLET-Mal 1DSLET [ 3H]EKC Control 1DSLET-Mal 1DSLET
B max (fmol/mg protein)
Kd (nM)
80.6 6 4.2 60.2 6 2.5 78.0 6 2.2
0.40 6 0.05 0.29 6 0.05 0.31 6 0.03
111.5 6 4.9 116.0 6 2.4 106.0 6 2.1
0.58 6 0.07 0.61 6 0.04 0.48 6 0.03
74.4 6 5.9 76.8 6 3.1 72.1 6 3.0
4.35 6 0.10 3.59 6 0.35 4.10 6 0.23
Note. Rats were intracerebroventricularly administered 40 nmol DSLET or DSLET-Mal. The control animals were treated in the same way with saline. The receptor binding experiments were carried out on whole brain homogenates.
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FIG. 4. [ 35S]GTPgS binding assays carried out on frontal cortex (top) and hippocampal (bottom) membranes of ICV-treated rats. Stimulation of [ 35S]GTPgS binding by different concentrations of [Ile 5,6]deltorphin II (h) and DAMGO (E) on control (filled symbols) and DSLET-Mal-treated (empty symbols) membranes is given as percentage of the non-stimulated (basal) level. Incubations were carried out for 60 min at 30°C. Non-specific binding was 45%, non-stimulated [ 35S]GTPgS binding was 71.9 6 19 fmol/mg protein for hippocampal and 75.2 6 18 fmol/mg protein in case of the frontal cortex membranes. Points represent means 6 S.E.M. from three independent experiments carried out in triplicates.
characteristics, compared to the parent compound, the enkephalin analogue DSLET. The maleoylated ligand showed high affinity to the [ 3H][Ile 5,6]deltorphin II labelled delta sites, having the equilibrium inhibition constant in the nanomolar range (Table 1). DSLETMal was moderately selective for delta receptors over mu receptors, which is consistent with the selectivity of the parent compound. In displacing [ 3H]naloxone, DSLET-Mal had a K i value of 49.6 nM, which increased more than 5-fold in the presence of sodium ions, suggesting its agonistic character. This result is supported by the [ 35S]GTPgS binding assays, where
TABLE 3
ED 50 Values Achieved in [ 35S]GTPgS Binding Assays in ICV-Treated Rat Brain Membranes Frontal cortex: ED 50 (nM)
[Ile 5,6]deltorphin II DAMGO
Hippocampus: ED 50 (nM)
Control
DSLET-Mal
Control
DSLET-Mal
22 199
3480 310
31 1030
.10000 1050
Note. Data represent the means of three individual experiments. Data were fitted with GraphPad Prism 2.01.
DSLET-Mal stimulated the binding with an ED 50 value of 9.5 nM. Since the inhibition of the specific binding of the used radioligands by DSLET-Mal remained despite several washes, it might be the result of an irreversible reaction. Examination of the concentration dependence of the irreversible effect revealed that higher concentration of the ligand is necessary for blocking the opioid receptors irreversibly, than it was expected from displacement studies. This is a general observation in case of in vitro measurements, the peptide affinity ligands (chloromethyl ketones (12, 29, 30), specific thiol reagents (31, 32)) show irreversibility in micromolar concentration range. The maleoyl moiety showed to be a very good reactive group, the percentage of irreversible binding (96% at 10 mM concentration) was higher than that of other reactive groups. As far as the selectivity of the irreversible binding is concerned, DSLETMal presented the same receptor preference as in the competition binding studies. As it is expected, in high concentrations (mM) the selectivity of the irreversible labelling decreased, the ligand bound to more of the available opioid receptors. The fact, that the pretreatment of the membrane with DSLET-Mal had effect on the number of binding sites (B max), but not on the K d of the binding, proves that the labelling of the receptor
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did not result in a conformational change, which would affect the free receptors’ functioning. The intracerebroventricular administration of the ligand reduced the density of the delta receptors, having no effect on the mu and kappa sites. Like in the case of in vitro pretreatment of the brain homogenate, the value of B max was reduced, no change in the K d value was observed (Table 2). According to the performed statistical analysis (unpaired t-test), the two-tailed p-value (0.0078) showed to be smaller than 0.05, which proved the significance in decrease at 95% of confidence interval. DSLET-Mal significantly decreased G protein activation by [Ile 5,6]deltorphin II, but had no effect on the DAMGO stimulated [ 35S]GTPgS binding 24 h after ICV administration in the studied brain regions. The effect of DSLET-Mal was not related to the distance of the regions from the ventricular space, since the frontal cortex was affected in the same manner as the hippocampus was. The effect of DSLET-Mal on [ 3H][Ile 5,6]deltorphin II binding was less than that found on [Ile 5,6]deltorphin II stimulated [ 35S]GTPgS binding. These results may be due to individual differences, since these two parameters were determined in different animals, as it is not possible to measure the receptor binding and receptor mediated stimulation of [ 35S]GTPgS binding using the brain of the same animal. Regardless to this, DSLET-Mal had qualitatively similar effects on both parameters, the results obtained with receptor binding are also observed at the signal transduction level. The results, that under in vivo conditions DSLET-Mal showed much higher selectivity than in the case of in vitro irreversible labelling can be explained by the differences in applied concentrations. While in in vitro conditions DSLET-Mal was present in 10 mM concentration, with intracerebroventricular administration a very small amount of DSLET-Mal reaches to the membranes. In these conditions, the labelling presents much higher selectivity. The structural requirements of ligands for irreversible attachment to receptors are quite specific. b-Funaltrexamine was proven to react with Lys 233 on the cloned rat mu opioid receptor (33). Knowing the structural similarity of the functional group in b-funaltrexamine and maleoyl group, DSLET-Mal is expected to be able to react with this function. Very precise sterical requirements are necessary, however, to attack the amino group on Lys, thus it is not surprising that no correlation can be shown between the receptor affinity (nanomolar range) and the irreversible binding (micromolar range) on the receptor. The other possibility for the maleoyl function is to react with the disulfide groups on the receptor. Among the affinity labels probed, the only reagents which proved to be effective below or in the micromolar range are the [D-Ala 2, Leu(CH 2S-3-nitro-2-pyridinesulphenyl]enkephalin derivatives (34) and [D-Ala 2, Leu 5, Cys 6]-
enkephalin (31). The morphiceptin analogs react at much higher concentration, although selectively (32). For the labelling, however, not only specific binding, but specific sterical arrangement is necessary. It seems that the binding site and the disulfide bond is not very close to each other, this is why in the case of shorter peptide, like morphiceptin, the labelling is not as effective as in the case of enkephalin derivatives. Further studies with DSLET-Mal may prove useful in understanding the mode of interaction of opioid peptides with their receptors and ligand-induced changes in G protein activation. Following radiolabelling DSLET-Mal is appropriate for in vitro autoradiography and gel fluorographic methods, which could provide further important results in localisation of binding sites, identifying the amino acid residues involved in the covalent binding. ACKNOWLEDGMENTS This study was made possible by financial supports from OTKA T 022104. The authors also thank helpful discussions with Dr. Krisztina Monory and Dr. Sa´ndor Benyhe.
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26. Bradford, M. M. (1986) Anal. Biochem. 72, 248 –254. 27. Leatherbarrow, R. J. (1992) GraFit version 3.0, pp. 1–287, Erithacus Software Ltd., Staines, UK. 28. Pert, C. B., and Snyder, S. H. (1974) Mol. Pharmacol. 10, 868 – 879. 29. Benyhe, S., Hepp, J., Szu˝cs, M., Simon, J., Borsodi, A., Medzihradszky, K., and Wollemann, M. (1986) Neuropeptides 8, 173–184. 30. Benyhe, S., Ketevan, A., Simon, J., Hepp, J., Medzihradszky, K., and Borsodi, A. (1997) Neuropeptides 31, 52–59. 31. Bowen, W. B., Hellewell, S. B., Kelemen, M., Huey, R., and Stewart, D. (1987) J. Biol. Chem. 262, 13434 –13439. 32. Motoyama, S., Takada, K., Yasunaga, T., Fujita, T., and Shimohigashi, Y. (1996) Bull. Chem. Soc. Jpn. 69, 3607–3612. 33. Chen, C., Yin, J., de Riel, J. K., DesJarlais, R. L., Raveglia, L. F., Zhu, J., and Liu-Chen, L.-Y. (1996) J. Biol. Chem. 271, 21422– 21429. 34. Shimohigashi, Y., Takada, K., Sakamoto, H., Matsumoto, H., Yasunaga, T., Kondo, M., and Ohno, M. (1992) J. Chromatogr. 597, 425– 428.
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Affinity Labeling of d Opioid Receptors by an EnkephalinDerivative Alkylating Agent, DSLET-Mal Ildiko´ Szatma´ri,* Gyo¨rgy Orosz,† Ka´lma´n Medzihradszky,† and Anna Borsodi* ,1 *Institute of Biochemistry, Biological Research Center and †Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Szeged, Hungary
Received October 4, 1999
Opioid binding properties of Tyr-D-Ser-Gly-Phe-LeuThr-NH-NH-Gly-Mal (DSLET-Mal), a novel enkephalinframed affinity label, was determined in rat brain membranes. In competition studies the ligand showed high affinity for the delta opioid sites, labelled by [ 3H][Ile 5,6]deltorphin II (K i 5 8 nM), whereas its binding to the mu ([ 3H]DAMGO) and kappa ([ 3H]EKC) sites was weaker. Preincubation of the rat brain membranes with DSLET-Mal at micromolar concentrations resulted in a wash-resistant and dose-dependent inhibition of the [ 3H][Ile 5,6]deltorphin II binding sites (96% blocking at 10 mM concentration). Intracerebroventricular (ICV) administration of DSLET-Mal reduced the density of delta opioid receptors and had no effect on mu and kappa receptors, as determined by saturation binding studies. [Ile 5,6]deltorphin II-stimulated [ 35S]GTPgS binding was determined in membrane preparations of different brain areas of the ICV-treated animals. In both frontal cortex and hippocampus DSLET-Mal significantly decreased G protein activation by the delta agonist, having no effect on DAMGO stimulated [ 35S]GTPgS binding. DSLET-Mal had qualitatively similar effects on both receptor binding and G protein activation. These characteristics of the compound studied suggest that DSLETMal can serve as an affinity label for further studies of the delta-opioid receptors. © 1999 Academic Press
Opioid receptors belong to the superfamily of G protein coupled receptors. They are integral membrane proteins characterised by amino acid sequences, which contain seven hydrophobic domains, representing the transmembrane regions. Opioid agonists stimulate the exchange of guanosine triphosphate (GTP) for guanosine diphosphate (GDP) on binding to their re1 To whom correspondence should be addressed at Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary. Fax: 36-62-433432. E-mail: [email protected].
ceptor by increasing the affinity of the Ga subunit of the G protein for GTP versus GDP. The GTP-Ga complex can than interact with intracellular effectors until an intrinsic GTPase on the Ga subunit hydrolyses GTP to GDP and the cycle repeats (1). The peptides Leu-enkephalin and Met-enkephalin are the naturally occurring ligands for the delta-opioid receptor (2). In efforts to produce more stable and selective ligands, several analogs of enkephalin have been synthesised such as: [D-Ala 2, D-Leu 5]enkephalin (DADLE) (3), [D-Thr 2, Leu 5, Thr 6]enkephalin (DTLET) (4) and [D-Ser 2, Leu 5, Thr 6]enkephalin (DSLET) (5). These peptides contain D-amino acids, the incorporation of which has been shown to stabilise peptides against proteolytic inactivation (6, 7). Opioid receptors have been shown to contain a thiol group at, or close to the ligand binding site (8, 9, 10). Alkylation of these sites by N-ethylmaleimid inhibits binding of mu and delta ligands, and the site is protected from alkylation by the bound ligand. The receptor thiol group is likely to be the nucleophile, which attacks receptor-specific, electrophilic affinity labels. Affinity ligands that form covalent bonds with receptors have been very useful in the elucidation of receptor structure (11). Many attempts have been made to prepare site directed irreversible probes for opioid receptors using alkaloid and peptide ligands. Alteration of free carboxyl termini of opioid peptides with functional groups resulted in ligands capable of interacting with the receptor in a wash-resistant manner. Peptides were modified with a number of reacting groups, including chloromethylketone (12, 13), melphalan (14, 15) and azido groups (16, 17). As thiol groups are known to easily react by addition to the maleoyl functional group, maleoyl group containing peptides were designed and tested in receptor binding studies. The maleoyl function is a highly reactive group, structurally it is very similar to the fumarate methyl ester, which was utilised in b-funaltrexamine (18), a widely utilised mu opioid irreversible ligand.
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The different maleoyl enkephalin analogues presented good selectivity to the delta opioid receptor and blocked a high percentage of these binding sites (19). In this study we determined the binding properties of the maleoylated DSLET in ligand binding and [ 35S]GTPgS binding assays. Furthermore, we investigated the effects of intracerebroventricular administration of this compound on agonist-stimulated [ 35S]GTPgS binding in rat brain membranes. As both frontal cortex and hippocampus are known to be enriched in delta opioid receptors (20), these two brain regions were selected for the functional assays. MATERIALS AND METHODS Chemicals. [ 3H]Ethylketocyclazocine (EKC; 20 Ci/mmol) was purchased from DuPont NEN (Cambridge, MA). [3H][D-Ala2(Me)Phe4Gly5ol]enkephalin (DAMGO; 55 Ci/mmol) was from Amersham, UK. [ 3H]naloxone (35 Ci/mmol) and [ 3H][Ile 5,6]deltorphin II (49 Ci/mmol) were synthesised in the Isotope Laboratory of Biological Research Center, Szeged (21, 22). All other chemicals used in this study were of analytical grade and purchased from Sigma (St. Louis, MO) or Reanal/Egis Pharmaceuticals (Budapest, Hungary). Synthesis of DSLET-Mal. Boc-Tyr(tBu)-D-Ser(tBu)-Gly-Phe-LeuThr(tBu)-OH was synthesized on 2-chlorotritylchloride resin (23) by Fmoc-technique. The details of the peptide synthesis is given elsewhere (24). The protected peptide acid was converted to its methyl ester by diazomethane and then reacted with hydrazine hydrate in methanol to obtain Boc-Tyr(tBu)-D-Ser(tBu)-Gly-Phe-Leu-Thr(tBu)NH-NH 2. This compound was reacted with maleoyl glycine in the presence of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate and diisopropyl-ethylamine in N,N-dimethyl formamide. The protecting groups were removed in trifluoroacetic acid containing 10% anisole. The crude product purified by preparative high performance liquid chromatography and analyzed by amino acid analysis and FAB-mass spectrometry. Intracerebroventricular (ICV) administration of peptides. Male Wistar rats (250 –300 g) were implanted with 20 G stainless steel canulae into the 3rd ventricle (1.3 mm rostral and 1.5 mm lateral from bregma, 3.5 mm ventral from the skull surface). Following 3– 4 days of recovery from surgery, animals were administered either 0 or 40 nmol of DSLET or DSLET-Mal in 4 ml of saline. Animals were sacrificed 24 h after the ICV administration, brain sections were removed and processed. Preparation of rat brain membranes. A crude membrane fraction was prepared from Wistar rat brains according to an earlier published method (25). The protein concentration was determined by the Bradford method (Bradford, 1986) (26), using bovine serum albumin as standard. Pretreatment of membranes (affinity labelling). Freshly prepared rat brain membranes (0.4 – 0.8 mg/ml) in 50 mM Tris-HCl (pH 7.4) buffer were preincubated with the compound to be tested at the concentrations indicated at 0°C for 1 h, in a final volume of 1 ml. Control tubes were run without the studied compound. After preincubation the mixtures were diluted to 1:15 with the same buffer and centrifuged (25000 g, 10 min). The pellets were resuspended in the buffer and incubated again for 15 min at 0°C. This washing step (centrifugation and resuspension) was repeated 4 times. The final pellets were resuspended in 4 ml of buffer and used immediately for [ 3H]naloxone binding assay in order to determine the amount of the irreversibly labelled binding sites. To determine the efficiency of the washing procedure, membranes were also preincubated with naloxone (reversible opioid ligand).
TABLE 1
Binding Affinities of DSLET and DSLET-Mal at Three Representative Opioid Ligand Binding Sites in Rat Brain Membranes K i (nM) Ligand
[ 3H]DAMGO
[ 3H][Ile 5,6]deltorphin II
[ 3H]EKC
DSLET DSLET-Mal
250 6 17 94 6 12
6.3 6 1.6 8.0 6 1.5
n.d. 147 6 24
Note. Rat brain membranes were incubated with the radioligands in the presence of the unlabelled peptide in 10 concentrations, ranging from 0.01 to 10000 nM. Assays were carried out as described under Materials and Methods. Values are averages of at least three experiments carried out in duplicate. n.d., not determined.
Receptor binding assay. The experiments were performed in glass for [ 3H]naloxone, [ 3H]EKC and [ 3H]DAMGO or plastic tubes for [ 3H][Ile 5,6]deltorphin II in 50 mM Tris-HCl buffer (pH 7.4) in a final volume of 1 ml and at a protein concentration of 0.3– 0.5 mg/ml. In case of [ 3H]EKC, the binding was performed in presence of 100 nM DAMGO and 100 nM [D-Ala 2, Leu 5]enkephalin in order to block the crossbinding to mu and delta sites. Incubation was started by addition of membrane suspension and terminated by rapid vacuum filtration through Whatman GF/C (for [ 3H][Ile 5,6]deltorphin II and [ 3H]DAMGO) or GF/B (in case of [ 3H]naloxone and [ 3H]EKC) filters using a Brandel Cell Harvester. After three washes with 5 ml portions of ice-cold buffer, the filters were dried at 37°C. The radioactivity was measured in a toluene based scintillation cocktail using a Wallac 1409 scintillation counter. The displacement curves were analyzed with the GraFit 3.0 using a nonlinear least squares algorithm for one site model. All experiments were carried out as duplicate assays and repeated at least three times. The given values are means 6 SEM. [ 35S]GTPgS binding. GTPgS is a thiol derivative of GTP that is resistant to hydrolysis by the GTPase activity of Ga. Opioid stimulation of [ 35S]GTPgS binding is an effective assay for G protein activation by opioids in vitro, using brain homogenates. Tubes containing 10 mg of protein, 30 mM GDP, 10 29–10 25 M opioid ligands, and 0.05 nM [ 35S]GTPgS, all in 50 mM Tris-HCl buffer containing 1 mM EGTA, 100 mM NaCl and 3 mM MgCl 2 in a final volume of 1 ml were incubated for 1 h, at 30°C. Non-stimulated activity was measured in the absence of tested compounds, non-specific binding was measured in the presence of 100 mM unlabelled GTPgS. The incubation was started by the addition of [ 35S]GTPgS and was terminated by filtrating the samples through Whatman GF/F glass fibre filters. Filters were washed three times with 5 ml of ice-cold 50 mM TrisHCl buffer (pH 7.4) in a Millipore filtration instrument, then dried. Bound radioactivity was measured in a Wallac 1409 scintillation counter using a toluene based scintillation cocktail. Stimulation is given as percent of the specific binding. Data were calculated from three independent experiments performed in triplicates.
RESULTS The ability of DSLET-Mal to compete reversibly with the binding of type specific opioid radioligands was determined over a wide concentration range of the studied ligand. The equilibrium inhibition constants (K i values) are shown in Table 1. DSLET-Mal bound with the highest affinity (K i 5 8.0 nM) to the
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FIG. 1. Inhibition of [ 3H][Ile 5,6]deltorphin II binding to rat brain membrane by various delta selective peptide ligands: (■) [Ile 5,6]deltorphin II, (Œ) [D-Ala 2, Leu 5]enkephalin, () DSLET, (F) DSLETMal, (p) DAMGO.
[ 3H][Ile 5,6]deltorphin II (delta) sites and had much lower potency in inhibiting both [ 3H]DAMGO (mu) and [ 3H]EKC (kappa) binding. Compared to the parent compound, the maleoylated peptide has the mu/delta selectivity ratio in the same range. Figure 1 shows the potency in inhibiting the [ 3H][Ile 5,6]deltorphin II binding of DSLET-Mal compared to other delta specific peptide ligands. As it is its affinity at the delta site was comparable to that of the other analogues. Sodium ions at 100 mM concentration decreased the affinity of DSLET-Mal suggesting its agonist character (28). The [ 35S]GTPgS binding assays showed its agonist character too: DSLET-Mal stimulated the GTPgS binding and the fact that addition of DSLET-Mal to [Ile 5,6]deltorphin II stimulated G protein activation had no effect on the reaction, excluded the antagonist property of the ligand. In order to test the irreversible binding of the studied ligand, rat brain membranes were prelabelled with
DSLET-Mal, then extensively washed to remove the reversibly bound ligand. Ligands producing a high degree of receptor occupation after this dissociation step are likely to be covalently attached. The irreversible binding of DSLET-Mal to rat brain membrane was tested with [ 3H][Ile 5,6]deltorphin II and compared to the effect of its parent compound DSLET. As shown in Fig. 2A, DSLET-Mal caused a dose-dependent, wash-resistant inhibition of the specific binding of [ 3H][Ile 5,6]deltorphin II. At 10 mM concentration a 96% irreversible blockade was observed. In the case of DSLET no such effect was observed. The receptortype selectivity of the irreversible binding was estimated using mu ([ 3H]DAMGO), delta ([ 3H][Ile 5,6]deltorphin II) and kappa ([ 3H]EKC) specific radioligands. Figure 2B shows the receptor types occupied in DSLET-Mal pretreated membranes. The effect of DSLET-Mal on receptor loss was moderately selective for delta receptors. Equilibrium saturation binding studies with [3H][Ile5,6]deltorphin II were also carried out with DSLET-Mal treated and control membranes (Fig. 3). Specific binding of [ 3H][Ile 5,6]deltorphin II was characterised by a single high affinity site. The maximal number of binding sites (B max) was substantially decreased in membranes preincubated with 10 mM affinity reagent. While in the case of the control membranes the B max showed to be 102.5 fmol/mg protein, on the pretreated membranes the value was just 19.4 fmol/mg. The equilibrium dissociation constant (K d) value of [ 3H][Ile 5,6]deltorphin II was not affected significantly by the presence of the maleoylated peptide, both in control and pretreated membranes K d presented values in subnanomolar range, 0.63 and 0.74 nM, respectively.
FIG. 2. Irreversible interaction of DSLET-Mal with different opioid receptor types. Rat brain membranes were preincubated with the respective peptide and then treated as described under Materials and Methods. Following this pretreatment the recovery of receptors was assessed using [ 3H]-ligands. Values are expressed in comparison to controls, which were preincubated without the peptide, but otherwise treated the same way. (A) The concentration dependence of irreversible interaction of the parent and maleoylated peptide with the delta opioid sites. (B) The receptor selectivity of the irreversible effect. Mu receptors were labelled with [ 3H]DAMGO (Œ), delta receptors were labelled with [ 3H][Ile 5,6]deltorphin II (■), and kappa receptors were labelled with [ 3H]EKC ().
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FIG. 3. Saturation binding isotherm (A) and Scatchard plot (B) of [ 3H][Ile 5,6]deltorphin II specific binding in control (■) and DSLET-Mal treated (h) membranes. In control experiments the membrane was preincubated without the peptide, but otherwise treated the same way.
In the next set of experiments we studied the effect of intracerebroventricular administration of DSLET-Mal on mu- and delta-agonist stimulated [ 35S]GTPgS binding. Firstly, equilibrium saturation binding studies with both [ 3H][Ile 5,6]deltorphin II, [ 3H]DAMGO and [ 3H]EKC were carried out in order to see the effect of DSLET-Mal on the density of the opioid receptor types. The intracerebroventricular administration of DSLETMal resulted in a decrease in the maximal number of binding sites available to the delta-specific radioligand, without significantly changing its affinity (K d). In order to see the significance of the decrease in the B max value, unpaired t-tests were performed. The obtained twotailed p value showed to be 0.0078. As shown in Table 2, the in vivo pretreatment did not significantly alter neither the affinity, nor the B max of the mu and kappa radioligands. The same experiments were carried out using the parent compound. The intracerebroventricular administration of DSLET had no effect neither on the maximal number of binding sites, nor on the equilibrium binding constant of the [ 3H][Ile 5,6]deltorphin II binding (Table 2). The mu- and delta-agonist stimulated [ 35S]GTPgS binding was carried out on the frontal cortici and hippocampi of control and ICV-treated animals. In both brain regions DSLET-Mal administration significantly decreased G protein activation by the delta agonist [Ile 5,6]deltorphin II, having no effect on DAMGOstimulated [ 35S]GTPgS binding (Fig. 4, Table 3). To see the irreversibility of the ICV administered compound, the membranes were washed as in the in vitro labelling experiments, and the same [ 35S]GTPgS binding experiments were carried out. The effect of DSLET-Mal remained after several washings. When the parent compound, DSLET was administered, no differences could be observed between the control and treated mem-
branes, neither in mu-, nor in delta-agonist stimulated [ 35S]GTPgS binding (data not shown). DISCUSSION A novel type of enkephalin-framed affinity label was prepared and tested for its activity both in vitro and in vivo. The potency of the binding of DSLET-Mal to the opioid receptors was investigated in competition experiments using crude membrane fraction of rat brain. The presence of the maleoyl group at the C terminus of the peptide did not change significantly the binding
TABLE 2
The Effect of Intracerebroventricular Administration of DSLET-Mal and DSLET on the Binding Constants (K d) and Maximal Binding Sites (B max) of Type-Specific Opioid Radioligands Ligand [ 3H][Ile 5,6]deltorphin II Control 1DSLET-Mal 1DSLET [ 3H]DAMGO Control 1DSLET-Mal 1DSLET [ 3H]EKC Control 1DSLET-Mal 1DSLET
B max (fmol/mg protein)
Kd (nM)
80.6 6 4.2 60.2 6 2.5 78.0 6 2.2
0.40 6 0.05 0.29 6 0.05 0.31 6 0.03
111.5 6 4.9 116.0 6 2.4 106.0 6 2.1
0.58 6 0.07 0.61 6 0.04 0.48 6 0.03
74.4 6 5.9 76.8 6 3.1 72.1 6 3.0
4.35 6 0.10 3.59 6 0.35 4.10 6 0.23
Note. Rats were intracerebroventricularly administered 40 nmol DSLET or DSLET-Mal. The control animals were treated in the same way with saline. The receptor binding experiments were carried out on whole brain homogenates.
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FIG. 4. [ 35S]GTPgS binding assays carried out on frontal cortex (top) and hippocampal (bottom) membranes of ICV-treated rats. Stimulation of [ 35S]GTPgS binding by different concentrations of [Ile 5,6]deltorphin II (h) and DAMGO (E) on control (filled symbols) and DSLET-Mal-treated (empty symbols) membranes is given as percentage of the non-stimulated (basal) level. Incubations were carried out for 60 min at 30°C. Non-specific binding was 45%, non-stimulated [ 35S]GTPgS binding was 71.9 6 19 fmol/mg protein for hippocampal and 75.2 6 18 fmol/mg protein in case of the frontal cortex membranes. Points represent means 6 S.E.M. from three independent experiments carried out in triplicates.
characteristics, compared to the parent compound, the enkephalin analogue DSLET. The maleoylated ligand showed high affinity to the [ 3H][Ile 5,6]deltorphin II labelled delta sites, having the equilibrium inhibition constant in the nanomolar range (Table 1). DSLETMal was moderately selective for delta receptors over mu receptors, which is consistent with the selectivity of the parent compound. In displacing [ 3H]naloxone, DSLET-Mal had a K i value of 49.6 nM, which increased more than 5-fold in the presence of sodium ions, suggesting its agonistic character. This result is supported by the [ 35S]GTPgS binding assays, where
TABLE 3
ED 50 Values Achieved in [ 35S]GTPgS Binding Assays in ICV-Treated Rat Brain Membranes Frontal cortex: ED 50 (nM)
[Ile 5,6]deltorphin II DAMGO
Hippocampus: ED 50 (nM)
Control
DSLET-Mal
Control
DSLET-Mal
22 199
3480 310
31 1030
.10000 1050
Note. Data represent the means of three individual experiments. Data were fitted with GraphPad Prism 2.01.
DSLET-Mal stimulated the binding with an ED 50 value of 9.5 nM. Since the inhibition of the specific binding of the used radioligands by DSLET-Mal remained despite several washes, it might be the result of an irreversible reaction. Examination of the concentration dependence of the irreversible effect revealed that higher concentration of the ligand is necessary for blocking the opioid receptors irreversibly, than it was expected from displacement studies. This is a general observation in case of in vitro measurements, the peptide affinity ligands (chloromethyl ketones (12, 29, 30), specific thiol reagents (31, 32)) show irreversibility in micromolar concentration range. The maleoyl moiety showed to be a very good reactive group, the percentage of irreversible binding (96% at 10 mM concentration) was higher than that of other reactive groups. As far as the selectivity of the irreversible binding is concerned, DSLETMal presented the same receptor preference as in the competition binding studies. As it is expected, in high concentrations (mM) the selectivity of the irreversible labelling decreased, the ligand bound to more of the available opioid receptors. The fact, that the pretreatment of the membrane with DSLET-Mal had effect on the number of binding sites (B max), but not on the K d of the binding, proves that the labelling of the receptor
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did not result in a conformational change, which would affect the free receptors’ functioning. The intracerebroventricular administration of the ligand reduced the density of the delta receptors, having no effect on the mu and kappa sites. Like in the case of in vitro pretreatment of the brain homogenate, the value of B max was reduced, no change in the K d value was observed (Table 2). According to the performed statistical analysis (unpaired t-test), the two-tailed p-value (0.0078) showed to be smaller than 0.05, which proved the significance in decrease at 95% of confidence interval. DSLET-Mal significantly decreased G protein activation by [Ile 5,6]deltorphin II, but had no effect on the DAMGO stimulated [ 35S]GTPgS binding 24 h after ICV administration in the studied brain regions. The effect of DSLET-Mal was not related to the distance of the regions from the ventricular space, since the frontal cortex was affected in the same manner as the hippocampus was. The effect of DSLET-Mal on [ 3H][Ile 5,6]deltorphin II binding was less than that found on [Ile 5,6]deltorphin II stimulated [ 35S]GTPgS binding. These results may be due to individual differences, since these two parameters were determined in different animals, as it is not possible to measure the receptor binding and receptor mediated stimulation of [ 35S]GTPgS binding using the brain of the same animal. Regardless to this, DSLET-Mal had qualitatively similar effects on both parameters, the results obtained with receptor binding are also observed at the signal transduction level. The results, that under in vivo conditions DSLET-Mal showed much higher selectivity than in the case of in vitro irreversible labelling can be explained by the differences in applied concentrations. While in in vitro conditions DSLET-Mal was present in 10 mM concentration, with intracerebroventricular administration a very small amount of DSLET-Mal reaches to the membranes. In these conditions, the labelling presents much higher selectivity. The structural requirements of ligands for irreversible attachment to receptors are quite specific. b-Funaltrexamine was proven to react with Lys 233 on the cloned rat mu opioid receptor (33). Knowing the structural similarity of the functional group in b-funaltrexamine and maleoyl group, DSLET-Mal is expected to be able to react with this function. Very precise sterical requirements are necessary, however, to attack the amino group on Lys, thus it is not surprising that no correlation can be shown between the receptor affinity (nanomolar range) and the irreversible binding (micromolar range) on the receptor. The other possibility for the maleoyl function is to react with the disulfide groups on the receptor. Among the affinity labels probed, the only reagents which proved to be effective below or in the micromolar range are the [D-Ala 2, Leu(CH 2S-3-nitro-2-pyridinesulphenyl]enkephalin derivatives (34) and [D-Ala 2, Leu 5, Cys 6]-
enkephalin (31). The morphiceptin analogs react at much higher concentration, although selectively (32). For the labelling, however, not only specific binding, but specific sterical arrangement is necessary. It seems that the binding site and the disulfide bond is not very close to each other, this is why in the case of shorter peptide, like morphiceptin, the labelling is not as effective as in the case of enkephalin derivatives. Further studies with DSLET-Mal may prove useful in understanding the mode of interaction of opioid peptides with their receptors and ligand-induced changes in G protein activation. Following radiolabelling DSLET-Mal is appropriate for in vitro autoradiography and gel fluorographic methods, which could provide further important results in localisation of binding sites, identifying the amino acid residues involved in the covalent binding. ACKNOWLEDGMENTS This study was made possible by financial supports from OTKA T 022104. The authors also thank helpful discussions with Dr. Krisztina Monory and Dr. Sa´ndor Benyhe.
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