Introduction

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Despite the sequence homology of the G protein-coupled ORL1 to the mu, delta and kappa opioid receptor, none of the opioid receptor ligands exhibited activity at ORL1 expressed in COS or CHO cells (Mollereau et al., 1994; Lachowicz et al., 1995), and a novel heptadecapeptide named orphanin FQ (Reinscheid et al., 1995) or nociceptin (Meunier et al., 1995) has been identified to be an endogenous ligand for ORL1. Nociceptin was found to specifically activate ORL1 expressed in cells (Meunier et al., 1995; Reinscheid et al., 1995, 1996; Butour et al., 1997) and a nociceptin receptor in neuroblastoma x glioma NG108-15 hybrid cells (Ma et al., 1997) and in membranes from mouse brain (Mathis et al., 1997) to inhibit the adenylate cyclase. However, nociceptin has been shown to bind to any of the opioid receptors only with extremely low affinity (Shimohigashi et al., 1996).

Furthermore, the pharmacological effects of nociceptin in various species differed from those of the opioids (reviewed by Henderson and McKnight, 1997). After isolation of nociceptin, a hyperalgesic response to the peptide when injected intracerebrovascularly into mouse was found (Reinscheid et al., 1995; Meunier et al., 1995). After further studies, however, the interpretation of the actions of nociceptin has become more complicated, especially when it was observed that intrathecally administered peptide produced an analgesic, rather than a hyperalgesic, response (see review by Henderson and McKnight, 1997) and that the acute hyperalgesia induced by intracerebrovascularly injected nociceptin was followed by a delayed analgesic response (Rossi et al., 1996). Nevertheless, it seems now to be clear that nociceptin supraspinally reverses opioid-mediated antinociception (Mogil et al., 1996; Tian et al., 1997) and produces persistent hyperalgesia when the analgesic effect of the endogenous opioids is blocked by opioid antagonists (Rossi et al., 1997).

Specific binding sites for nociceptin, thought to mediate the actions of the peptide in brain, have been found in membranes obtained from rat (Dooley and Houghten, 1996; Makman et al., 1997; Ardati et al., 1997), guinea pig (Shimohigashi et al., 1996) and mouse brain (Mathis et al., 1997) as well as from neuroblastoma x glioma NG108-15 hybrid cells (Ma et al., 1997). Remarkably, their affinities reported differed widely (K_d 0.004-5.0 nM), which was even true for those reported (K_d 0.021-1.2 nM) for ORL1 expressed in CHO (Reinscheid et al., 1995, 1996; Ardati et al., 1997; Butour et al., 1997; Fukuda et al., 1997) or HEK cells (Ardati et al., 1997; Shimohigashi et al., 1996). The presence of more than one receptor affinity state for agonist binding to the nociceptin receptor might indicate different functional interaction of the G protein-coupled receptor with the signal-transducing G proteins.

The aim of this study was, therefore, to investigate G protein activation by the activated nociceptin receptor in rat brain. In membrane preparations, such an activation process is frequently monitored by studying the agonist stimulation of high-affinity GTPase activity of the G protein -subunit or the shift in receptor binding affinity by a stable GTP analog. Whereas GTPase activity reflects only the steady-state kinetics of the overall G protein activity cycle, the shift in affinity by GTP is rather variable and sometimes very small in size. Therefore, to get insights in the process of coupling of the nociceptin receptor to the G proteins in a more quantitative manner, we characterized the binding of the radiolabeled GTP analog ³⁵S-GTPS to the G proteins within the rat brain membranes during stimulation by nociceptin agonists. As a result we found that in rat brain a single high-affinity nociceptin binding site is coupled to low-potency stimulation of GTP binding.

	Methods and Materials

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Substances and buffer. Nociceptin [nociceptin(1-17)], its fragments nociceptin(1-13) and nociceptin(1-9), and its analog D-Ala⁷-nociceptin(1-17) were synthesized in our institute (Berlin). H-Tyr¹⁴-nociceptin (45 Ci/mmol), ¹²⁵I-Tyr¹⁴-nociceptin (2200 Ci/mmol) and ³⁵S-GTPS (1208 Ci/mmol) were obtained from NEN (Boston, MA). Gpp(NH)p, GTPS, GDP, naltrindole, U-69593, DAMGO and naloxone were from Sigma (Delsenhofen, Germany). A total of 50 mM Tris/HCl, pH 7.4, containing 3 mM MgCl₂ and 0.2 mM EGTA with additions as specified was used as buffer in all experiments.

Nociceptin binding to rat cerebral cortex membranes. The cerebral cortex of male Wistar rats (about 250 g) was homogenized with an Ultra-Turrax in buffer, and the total membrane fraction was obtained by centrifugation. The receptor binding experiments were performed as described earlier for the mu opioid receptor (Albrecht et al., 1997). Briefly, about 250 µg of membrane protein in buffer containing 0.1% BSA and the protease inhibitors aprotinin (0.0015%) and bacitracin (0.15 mM) was incubated with increasing concentrations of ³H-Tyr¹⁴-nociceptin (saturation experiment) or with the constant concentration of about 50 pM ³H-Tyr¹⁴-nociceptin and 0.001 to 10,000 nM unlabeled nociceptin (displacement experiment) in a total volume of 1 ml at 30°C for 90 min. In the displacement experiments, nonspecific binding in presence of 10 to 1000 nM unlabeled nociceptin was as low as <2%. For the parameters of nociceptin receptor binding to be compared with those of receptor-stimulated GTPS binding, in some binding experiments 100 mM NaCl, 10 µM Gpp(NH)p or GTPS and 20 µM GDP each alone or in combination were added to the incubations. These substances were included in the experiments on nociceptin-stimulated GTPS binding. Furthermore, in one series of three experiments, displacement curves of the binding of ³H-Tyr¹⁴-nociceptin using nociceptin, nociceptin(1-13), nociceptin(1-9) and D-Ala⁷-nociceptin(1-17) were obtained from incubations in presence of 100 mM NaCl/20 µM GDP at 25°C for 2 hr. The samples were filtered through Whatman GF/B filters using a Brandel-Harvester, and the filters were counted for ³H-activity. The receptor binding parameters, i.e., the dissociation constant K_d and the binding capacity B_max, were estimated using the program RADLIG 4.0 (BIOSOFT, Cambridge, UK). The displacement by nociceptin of the binding of the iodinated tracer peptide ¹²⁵I-Tyr¹⁴-nociceptin was compared once with that of the ³H-labeled tracer. Using ³H-labeled nociceptin at concentrations of as low as 40 pM in the displacement studies and in the beginning part of the tracer saturation binding curve (fig. 1) resulted in a maximum specific binding of about 50% of the added tracer amount. These unusual conditions were necessary for obtaining the parameters of the high-affinity binding, and the program RADLIG 4.0 is able to estimate the binding parameters at sufficient accuracy even under such conditions.

Nociceptin binding in coronal sections of rat cerebral cortex. Rats were killed by decapitation. Their brains and spinal cords were rapidly removed, frozen on dry ice and stored at 70°C until use. Coronal sections (10 µm) of both tissues were cut on a cryostat (CM 3000 Leica), thaw-mounted onto gelatin-coated slides and stored at -70°C until further processing. Sections of frontal cortex were preincubated in buffer at 25°C for 15 min. After washing two times for 30 sec with buffer, the sections were incubated with 50 pM ¹²⁵I-Tyr¹⁴-nociceptin and different concentrations of unlabeled nociceptin (0.001 up to 5 nM; displacement experiment) at 25°C for 2 hr in buffer containing 0.0015% aprotinin, 0.15 mM bacitracin and 0.1% BSA. After this time, the slides were washed with chilled 50 mM Tris/HCl and deionized water, dried and, together with autoradiographic I-micro-scales (Amersham, Braunschweig, Germany), exposed to UR-imaging plates (FUJI Photo Film Co., Tokyo, Japan) for 16 hr. The plates were scanned and analyzed using the bio-imaging analysis system BAS-3000 (FUJI) linked to the micro computer imaging device system from Imaging Research Inc. (St. Catherines, Canada). When the binding of 0.01 to 100 nM ³H-Tyr¹⁴-nociceptin was determined (saturation experiment), TR-plates (FUJI) were used for the exposition of the slides for at least 60 hr. The binding capacity was quantified by ³H-micro-scales (Amersham), and the binding parameters were determined as described above.

Nociceptin-stimulated ³⁵S-GTPS binding to rat cerebral cortex membranes. A total of 10 to 20 µg of membrane protein prepared from rat cortex as described above was incubated with 50 pM ³⁵S-GTPS in presence or absence of nociceptin at 30°C for the indicated times in a total volume of 1 ml of buffer supplemented with 20 µM GDP and 100 mM NaCl. The reaction was terminated by filtration through Whatman GF/B filters using a Brandel-Harvester, and the filters were counted for ³⁵S-activity. Basal, nonspecific and nociceptin-stimulated binding of ³⁵S-GTPS were defined as binding of the tracer in absence of nociceptin, in presence of 10 µM unlabeled GTPS, and as difference of binding in presence and absence of nociceptin, respectively. The affinity and capacity of the basal and nociceptin-stimulated binding of GTPS were estimated from the displacement of the binding of ³⁵S-GTPS by unlabeled GTPS as described for receptor binding. EC₅₀ values for the stimulation of ³⁵S-GTPS binding by nociceptin were calculated from concentration-response curves by a four-parameter logistic curve fitting program. Additionally, nociceptin concentration-response curves were compared with those of the shortened sequences nociceptin(1-13) and nociceptin(1-9) and the analog D-Ala⁷-nociceptin at 25°C.

Nociceptin- and DAMGO-stimulated ³⁵S-GTPS binding in coronal sections of rat brain and spinal cord. ³⁵S-GTPS binding to slide-mounted sections obtained from rat brain and spinal cord as described above was studied essentially under conditions as recently described in literature (Sim et al., 1995, 1996a). The sections were incubated in buffer containing 100 mM NaCl for 10 min at 25°C followed by incubation in the same buffer containing additionally 2 mM GDP for 15 min. Then they were incubated in this medium for 2 hr at 25°C with 50 pM ³⁵S-GTPS in presence and absence of 3 µM nociceptin or 3 µM DAMGO to obtain maximal stimulation of the binding of ³⁵S-GTPS compared to basal binding. After rinsing in cold buffer and deionized water, the slides were dried and, together with C-microscales (Amersham) for quantification, exposed to UR-imaging plates for 16 hr. The plates were scanned and analyzed using the BAS-micro computer imaging device combination. We confirmed that the concentration of GDP as high as 2 mM was necessary to suppress the basal binding of ³⁵S-GTPS to an extent that allowed maximum stimulation of binding of the nucleotide by the ligands over basal binding (Sim et al., 1995), in contrast to the binding to membranes, which was studied at much lower concentrations of GDP.

	Results

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Receptor binding of nociceptin. At 30°C the binding of 0.15 and 1.5 nM ³H-Tyr¹⁴-nociceptin to membranes from the rat cortex reached equilibrium after 60 min, was stable until 150 min and increased linearly with the concentration of membrane protein (200-1300 µg in 1 ml incubate). In presence of 100 mM NaCl/20 µM GDP, i.e., the medium used for studies on binding of ³⁵S-GTP S, binding equilibrium was also reached within 60 min.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Total, specific, and nonspecific binding (curves 1, 2 and 3, respectively) of 3H-Tyr14-nociceptin to rat cerebral cortex membranes in Tris buffer at 30°C, fitted to a one-site binding model, and displacement of bound 3H-Tyr14-nociceptin by unlabeled nociceptin in the absence (curve 4) and presence of 10 µM Gpp(NH)p (curve 5) or 100 mM NaCl (curve 6). Bars represent S.D. values for the experimentally determined bound tracer amounts and the calculated free tracer concentrations.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Displacement of 125I-Tyr14-nociceptin binding in coronal sections of rat frontal cerebral cortex by nociceptin, obtained from incubations of the sections with 50 pM tracer in Tris buffer for 2 hr at 25°C in three independent experiments (, , ). Each point represents the mean value obtained from 10 sections.

Nociceptin-stimulated binding of GTPS. In absence of any peptide ligand, the basal binding of ³⁵S-GTPS at 50 pM concentration to rat cortex membranes was increased linearly with protein concentrations of 2 to 20 µg per assay tube. This binding was nearly totally displaced by 10 µM unlabeled GTPS (nonspecific binding <5% of basal binding). Nociceptin dose-dependently (1-3000 nM) stimulated the binding (fig. 3) with an apparent EC₅₀ value of 9.11 ± 1.00 (S.E.) nM. At 1 µM, it maximally stimulated the binding 2.73 ± 0.17-fold (mean ± S.D.) over basal binding independent of the incubation time of 5 to 240 min (fig. 4). During this long time, neither basal binding nor nociceptin-stimulated binding reached equilibrium. However, due to their proportional increase the stimulation factor obtained with the peptide remained constant with time (fig. 4).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Concentration-response curve for the stimulation of binding of 50 pM 35S-GTPS to rat cerebral cortex membranes by nociceptin in Tris buffer/20 µM GDP/100 mM NaCl for 60 min at 30°C (mean ± S.D. of fmol/mg protein). Dashed line: Fitted curve of nociceptin binding to the membranes under identical conditions (compare fig. 1).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Time-dependency of the 35S-GTPS binding (mean ± S.D.) to rat cerebral cortex membranes in Tris buffer/20 µM GDP/100 mM NaCl with 50 pM tracer at 30°C in absence (basal binding, ) or in presence of 1 µM nociceptin (), and in presence of 10 µM unlabeled GTPS (nonspecific binding, ). Basal binding was subtracted from binding in presence of nociceptin to obtain the amount of stimulated binding (), and binding in presence of nociceptin was divided by basal binding, each corrected for the nonspecific binding, to obtain the stimulation factor (asterisk).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Comparison of the displacement of the binding of 3H-Tyr14-nociceptin to rat cerebral cortex membranes by nociceptin (), nociceptin(1-13) (), D-Ala7-nociceptin(1-17) (), and nociceptin(1-9) () in incubations in Tris buffer/20 µM GDP/100 mM NaCl for 2 hr at 25°C (upper panel) with the concentration-response curves for 35S-GTPS binding to the membranes (lower panel) stimulated by the same peptides under identical conditions (data representative of three experiments).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Displacement of binding of 50 pM 35S-GTPS (mean ± S.D., bars within symbols) to rat cerebral cortex membranes by unlabeled GTPS in absence () and presence () of 1 µM nociceptin in Tris buffer/20 µM GDP/100 mM NaCl for 240 min at 30°C. Nociceptin-stimulated binding () was calculated as difference between binding in presence and absence of nociceptin.

View larger version (38K): [in this window] [in a new window]   Fig. 7.   Comparison of basal, DAMGO- and nociceptin-stimulated 35S-GTPS binding in adjacent coronal sections obtained from different rat central nervous system areas (frontal, cingulate and occipital cortex, caudate putamen, amygdala, thalamus, hypothalamus, periaqueductal gray, cervical and thoracal spinal cord). The sections were incubated in Tris buffer/2 mM GDP/100 mM NaCl with 50 pM 35S-GTPS for 2 hr at 25°C. Binding is expressed as dpm/mg wet weight (mean ± S.D.).

	Discussion

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In this study, rat cerebral cortex was used to investigate the binding of nociceptin to its receptor and the coupling of the receptor to G proteins through nociceptin-stimulated binding of GTPS, because with brain sections it was found that, of all central nervous system regions studied, rat cortex showed the highest factor for the stimulation of ³⁵S-GTPS binding by the peptide over basal binding (fig. 7). Using membranes and sections of rat cortex, two labeled nociceptin peptides, H-Tyr¹⁴-nociceptin and ¹²⁵I-Tyr¹⁴-nociceptin, and two types of binding studies (saturation, displacement), one single high-affinity and high-capacity binding site for nociceptin in rat brain was found (figs. 1 and 2), the K_d value ranging from 21.6 to 116.7 pM, dependent on the preparation and kind of method used. This high affinity essentially agreed with that found for the nociceptin receptor in brain membranes from guinea pig (K_d 16 pM, Shimohigashi et al., 1996), mouse (K_d 100 pM, Mathis et al., 1997), and rat (K_d 50-100 pM, Ardati et al., 1997; Makman et al., 1997) and in CHO cells (K_d 50-190 pM, Reinscheid et al., 1995, 1996; Ardati et al., 1997; Butour et al., 1997). However, it contrasted with the much lower affinities found for NG108-15 cells (Ma et al., 1997), rat brain membranes (Dooley and Houghten, 1996), CHO cells (Fukuda et al., 1997) and HEK cells (Shimohigashi et al., 1996) with K_d values between 1 and 5 nM. Therefore, displacement of the binding of 1.5 nM ³H-Tyr¹⁴-nociceptin to rat cortex membranes was performed (0.1-1000 nM unlabeled nociceptin), resulting in an apparent displacement curve with an IC₅₀ value of about 2 nM (data not shown). However, the detailed analysis of the curve showed that it represented exactly isotopic dilution of the tracer binding to a site already fully saturated at 1.5 nM labeled peptide. Therefore we did not find any evidence for a second binding site with a K_d value around 1 nM in rat cortex, in contrast to the findings in mouse brain (Mathis et al., 1997). Different temperatures as used here and in various studies (30°C/25°C) cannot be responsible for the discrepancies in the affinities observed because we found the K_d values at 25°C and 30°C to differ not more than by 10% (data not shown).

Before the identification of nociceptin as endogenous ligand for the ORL1 receptor, a lot of studies had already shown that none of the selective ligands for the mu, delta and kappa opioid receptor activated the ORL1 despite the exceptionally high structural homology among these receptors (reviewed in Reinscheid et al., 1995; Meunier, et al., 1995). In agreement with this fact, naloxone and selective opioid ligands did not compete with nociceptin for the binding sites in rat cortex at all. However, as the homology profile of the receptors would indicate, the ORL1 exhibits signaling mechanisms similar to the opioid receptors. Activation of the receptor was found to inhibit adenylate cyclase, indicative of its coupling to Gi-proteins (Reinscheid et al., 1995, 1996; Meunier et al., 1995; Mathis et al., 1997; Ma et al., 1997; Butour et al., 1997). Further evidence is given by our findings that the stable GTP analogs Gpp(NH)p and GTPS as well as NaCl decreased the nociceptin receptor affinity in rat cortex (fig. 1), typical of opioid receptors. However, the shift was only moderate (2- to 3-fold) and not additive. This contrasts with results on ORL1 expressed in CHO cells (Butour et al., 1997), where NaCl increased the capacity of a preexisting very low-affinity nociceptin binding site, and where the shift was further increased by Gpp(NH)p. Furthermore, Gpp(NH)p drastically shifted the proportion of high to low affinity sites in HEK cells, but at the same time only moderately the affinity of a single site in CHO cells (Ardati et al., 1997). These differences show that the existence of a low-affinity binding site and the exact coupling of the receptor to the G proteins may depend on the type of cell expressing the receptor and on its expression level (Ardati et al., 1997), and that conclusions from the situation in cells expressing the receptor to that in native tissues have to be made cautiously.

The coupling of the nociceptin receptor to G proteins was directly observed by the stimulation of the binding of ³⁵S-GTPS in the membranes of cortex (fig. 3) as well as in sections from brain and spinal cord (fig. 7). Both basal and nociceptin-stimulated binding of the labeled nucleotide to the membranes proceeded at the same very slow rate, not reaching equilibrium even after incubation for 4 hr at 30°C (fig. 4). Obviously, the kinetics of both kinds of binding as seen in figure 4 is mostly dictated by the dissociation of GDP, required in the incubations to inactivate the G proteins and thus providing low basal levels of binding, from the GDP/GTP binding site of the G protein in the GDP-GTP exchange reaction (Wieland and Jakobs, 1994). Such an explanation should only be true if the basal binding of GTPS observed was binding to a specific site and not just nonspecific. With this in line is the fact that not only the stimulated but also the basal binding sites for GTPS were found to be specific with K_d values of 1.57 and 45.8 nM, respectively (fig. 6), but with the capacity of the basal binding sites being 15-fold higher than that of the nociceptin-stimulated binding sites.

The specific nature of the basal binding of GTPS might be interpreted as basal coupling of unoccupied receptors to G proteins. Then, the manifold higher capacity of basal over nociceptin-stimulated GTP binding sites found here would reflect constitutive activities of a lot of receptors. However, this seems to be unlikely due to the differences in affinity between basal and receptor-stimulated GTP binding, as seen here for the nociceptin receptor, characterizing basal and stimulated GTP binding sites as different states. More likely, basal binding of GTPS may be assumed to be binding to the nucleotide site in the G subunit within the heterotrimeric G protein in equilibrium with GDP, as opposed to binding to G dissociated from the G dimer after stimulation by receptor occupancy. Conclusively, in a native cell the nucleotide binding site in the heterotrimeric G protein may be occupied not only by GDP but also partly by GTP.

The GTPS binding isotherms could be evaluated according to the law of mass action, and from the parameters calculated it is concluded that nociceptin stimulated the binding of GTPS to G proteins by decreasing the K_d from K_{dlow aff.} 45.8 nM to K_{dhigh aff.} 1.57 nM, i.e., by increasing the affinity of the GTP binding site by 29-fold. This shift is higher than the about 3-fold increase in the binding affinity of GTPS by µ-opioid receptor activation in rat striatal membranes (Sim et al., 1996b). According to the law of mass action, if a binding site changes from low to high affinity, the amount of bound ligand in equilibrium with the free ligand concentration  is increased by the factor q = (+K_{dlow aff.)})/(+K_{dhigh aff.}). With the free ³⁵S-GTPS concentration being about 50 pM in the stimulation experiments, for the nociceptin receptor-coupled G proteins the amount of ³⁵S-GTPS binding stimulated by nociceptin over basal binding is calculated to be 28-fold. However, the maximum stimulation in the bound amount observed, i.e., the quotient of ³⁵S-GTPS binding in presence and absence of 1 µM nociceptin, was only 2.7 (fig. 4). Taking the 15-fold excess of the total basal (B_max 46.56 pmol/mg) over stimulated binding sites (B_max 3.03 pmol/mg) into account, it was calculated that a measurable factor of only 2-3 could be obtained as was the case (fig. 4). Generally, a sufficient part of G proteins involved in activation, a high increase in their affinity to GTP, and a low basal binding of GTP to the total of all G proteins will enable the stimulation of binding of a GTP analog to G proteins to be observed in any receptor activation in the tissue or cell under study.

In contrast to the parameters of the binding of nociceptin to its receptor, those of GTPS were not obtained at equilibrium (fig. 4) and are, therefore, only apparent parameters. The K_d values for the basal and nociceptin-stimulated binding of GTPS and especially their relation, however, should be rather robust due to the factor of stimulated over basal binding being unchanged with time (fig. 4). The binding capacities estimated, however, are not correct in absolute terms and are only meaningful when compared under identical conditions. From the capacity of the nociceptin receptor (290 fmol/mg) and of the nociceptin-stimulated GTPS binding sites after incubation for 4 hr (3 pmol/mg) it is concluded that one receptor site is able to stimulate at least 10 G protein sites for GTP.

Ligand binding by a G protein-coupled receptor is immediately coupled to the stimulation of the G proteins by stimulating the binding of GTP. Therefore, the main problem in the interpretation of the data on GTP binding lies in the low potency of nociceptin in stimulating the GTPS binding in cortex membranes (fig. 3) as compared with its high affinity to its receptor (fig. 1). As with membranes, sections from the cortex exhibited high-affinity receptors (fig. 2), but only at 0.1 to 1 µM concentrations of nociceptin was saturation of binding of ³⁵S-GTPS reached. Nociceptin stimulated the nucleotide binding in membranes with an EC₅₀ of 9.11 nM, which compares well with 19.8 nM as found in another study (Sim et al., 1996a), but which was much higher than the K_d of 0.03 nM for receptor binding found here. Generally, when G proteins are activated by agonist-stimulated binding of GTP, the affinity of the receptors is lowered. This was also observed in this study for the nociceptin receptor but the decrease of the K_d by <3-fold by the GTP analogs Gpp(NH)p (fig. 1) or GTPS was rather moderate. The same was true for the influence of NaCl (fig. 1), which was included in the GTP binding assays. Furthermore, 20 µM GDP, also included in the assay for lowering basal binding of ³⁵S-GTPS, had no influence on the nociceptin receptor affinity in absence or presence of NaCl. Remarkably, GDP prevented the receptor affinity shift by 10 µM GTPS, although under this condition the nucleotide binding site was fully occupied by GTPS (K_d 1.57 nM). Therefore, not competition by GDP but an allosteric influence of it on the GTPS-induced receptor affinity shift may be responsible for this observation.

In conclusion, under identical experimental conditions the affinity of nociceptin for its receptor was about 100-fold higher than its potency in stimulating the GTP binding. This would mean that about 90% of the concentration-response curve was accomplished between 90 to 100% occupancy of the receptor (fig. 3) or that the stimulation was mediated through a low-affinity site or state not detectable in the binding studies. When the specificity of the coupling between receptor binding and ³⁵S-GTPS binding was studied, the order of magnitude of the receptor affinities of nociceptin, nociceptin(1-13), D-Ala⁷-nociceptin, and nociceptin(1-9) (K_d 1:7.9:36.7:>10,000) was nearly the same (fig. 5) as that of their potencies in stimulating the S-GTPS binding (EC₅₀ 1:4.5:30:not measurable). Such was also the specificity seen in the inhibition of cAMP accumulation in CHO cells stably expressing the ORL1-receptor (Reinscheid et al., 1996). Remarkably, whereas D-Ala⁷-nociceptin was a partial agonist in inhibiting cAMP accumulation (Reinscheid et al., 1996) it showed in this study full maximum activity in stimulating the GTPS binding. In agreement with an earlier report (Sim et al., 1996a), no stimulation of nucleotide binding was identified in the caudate putamen, an area in which the mu opioid agonist DAMGO highly stimulated the binding (fig. 7). This parallels the very low levels of ORL1 immunoreactivity in this region, in contrast to other regions (Anton et al., 1996) that also expressed stimulation of GTP binding (fig. 7). Taken together, the specificity of the nociceptin-stimulated binding of GTPS corresponded to the properties of the high-affinity nociceptin receptor identified in rat brain.

In summary, our data show for the first time that a high-affinity nociceptin receptor in rat brain is coupled to a comparably very low-potency stimulation of GTPS binding. The exact mechanism of this coupling remains open. Because of the specificity of the stimulation of GTPS binding corresponding to that of the high-affinity receptors, it appears to be unlikely that an independent second low-affinity receptor site is responsible for the coupling. Rather, it seems reasonable to suggest the involvement of a low-affinity state of the receptor. Several lines of evidence have indicated the existence of multiple forms of a receptor complex (Gudermann et al., 1996), including opioid receptors (Werling et al., 1988; Wong et al., 1994), at any one time. Therefore, one explanation for our results on the nociceptin receptor would be that a small part of agonist-occupied receptors is switched to a low-affinity state, not detectable in binding studies in the presence of an excess of sites in high-affinity state. Because of the high capacity of the nociceptin receptors and of nociceptin-stimulated GTP binding sites, even a small part of the receptors in low-affinity state in equilibrium with the high-affinity state and stabilized at high agonist concentrations could provide enough receptor sites that catalytically stimulate GTP binding.