Introduction

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Cannabinoid receptors of the CB1 type mediate the central nervous system actions of cannabimimetic compounds (Dewey 1986; Martin 1986). CB1 receptors belong to the superfamily of seven transmembrane-spanning domain, G-protein-coupled receptors (Matsuda et al., 1990) and are negatively coupled to adenylyl cyclase (Howlett, 1984) and Ca⁺⁺ channels (Mackie and Hille, 1992; Mackie et al., 1995) and positively coupled to K⁺ channels (Hampson et al., 1995; Mackie et al., 1995) via pertussis-toxin sensitive (G_i/o) G-proteins (Howlett and Fleming, 1984; Howlett, 1985; Howlett et al., 1986; Bidaut-Russell et al., 1990). Two splice variants of CB1 mRNA (CB1 and CB1A) have been identified in human and rat brain (Shire et al., 1995). CB1 receptors are widely distributed with relatively high density in mammalian brain (Herkenham et al., 1991b; Jansen et al., 1992). Cannabinoid effects on memory, body temperature and motor function (Dewey, 1986) are consistent with the distribution of CB1 receptors in the hippocampus, hypothalamus and basal ganglia (Herkenham et al., 1991b; Jansen et al., 1992; Sim et al., 1995), yet the signal transduction mechanisms which mediate these biological actions are not well characterized.

For G-protein-coupled receptors such as CB1, the initial step in the signal transduction cascade which determines agonist efficacy is the activation of the G-protein (Kenakin, 1993). This step can be measured effectively by use of net agonist-stimulated [³⁵S]GTPS binding in the presence of excess GDP (Hilf et al., 1989; Offermanns et al., 1991; Lorenzen et al., 1993; Sim et al., 1995; Traynor and Nahorski, 1995; Selley et al., 1996). Agonist-stimulated [³⁵S]GTPS autoradiography and membrane binding assays are useful in determining relative levels of G-protein activation by agonists acting through a given G-protein-coupled receptor. When the apparent B_max of [³⁵S]GTPS binding and B_max of receptor binding are compared, a receptor/transducer amplification factor can be calculated as the relative number G-proteins activated on a per receptor molecule basis (Gierschik et al., 1991; Sim et al., 1996c). With such analyses, we recently demonstrated in rat striatal membranes that cannabinoid receptors are less efficiently coupled to G-proteins when compared with opioid receptors. In those studies, cannabinoid receptors activated only twice as many G-proteins as mu and delta opioid receptors, despite the 10-fold greater abundance of cannabinoid receptor binding sites than either type of opioid receptor. Therefore, each cannabinoid receptor activated only one seventh as many G-proteins as each mu or delta opioid receptor (Sim et al., 1996c).

The choice of radioligand used for receptor binding assays is critical. In our previous studies, amplification factors were calculated using high-affinity agonist ([³H]WIN 55212-2) binding (Sim et al., 1996c). However, such binding cannot be completely correlated with agonist-activated G-proteins because high-affinity agonist binding cannot be measured under the same assay conditions (with sodium and GDP) (Devane et al., 1988) as agonist-stimulated [³⁵S]GTPS binding. One of the important goals of the present study was to compare values of amplification factors calculated by high-affinity agonist receptor binding with those calculated from total receptor binding (determined with ³H-labeled antagonist). The recent synthesis of SR141716A as a selective CB1 antagonist (Rinaldi-Carmona et al., 1994), and the development of [³H]SR141716A as an antagonist radioligand for CB1 receptors (Rinaldi-Carmona et al., 1996), allowed for this opportunity, because [³H]SR141716A binding is unaffected by sodium, magnesium or guanine nucleotides (Rinaldi-Carmona et al., 1996).

Previous autoradiographic analysis of rat brain sections has revealed that cannabinoid receptors (determined by [³H]WIN 55212-2 binding) and cannabinoid activation of G-proteins (determined by net WIN 55212-2-stimulated [³⁵S]GTPS) have similar distributions (Sim et al., 1995; Childers and Deadwyler, 1996). However, some differences were observed: some regions with relatively low receptor density exhibited high levels of G-protein activation, whereas other regions with similar receptor densities exhibited low levels of G-protein activation. To further investigate this observation quantitatively, the present study compared saturation binding analyses of cannabinoid receptor-stimulated [³⁵S]GTPS binding and receptor binding in membrane homogenates from 10 different rat brain regions. [³⁵S]GTPS binding was determined in the presence of maximally effective concentrations of WIN 55212-2, and receptor density and affinity were measured with both the agonist, [³H]WIN 55212-2, and the antagonist, [³H]SR141716A. These results confirm that the efficiency of cannabinoid receptor coupling to G-proteins differs throughout the brain. In this study, the receptor/transducer amplification factor is defined as the apparent B_max of net agonist-stimulated [³⁵S]GTPS binding divided by the B_max of cannabinoid receptor ligand binding sites. The fraction of cannabinoid agonist high-affinity binding sites is defined as the B_max of ³H-labeled agonist ([³H]WIN 55212-2) binding divided by the B_max of ³H-labeled antagonist ([³H]SR141716A) binding.

	Materials and Methods

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Materials. Male Sprague-Dawley rats were purchased from Zivic Miller (Zeleinople, PA). [³⁵S]GTPS (1000-1250 Ci/mmol), [³H]WIN 55212-2 (45.5 Ci/mmol) and Reflections^TM film were purchased from New England Nuclear Corp. (Boston, MA). [³H]SR141716A (43-65 Ci/mmol) and Hyperfilm max were obtained from Amersham Life Sciences (Arlington Heights, IL). WIN 55212-2 was purchased from Research Biochemicals International (Natick, MA). SR141716A was a generous gift from Dr. Francis Barth at Sanofi Recherche (Montpellier, France). GDP for membrane [³⁵S]GTPS binding assays and GTPS were purchased from Boehringer Mannheim (New York, NY). GDP for [³⁵S]GTPS autoradiography was purchased from Sigma Chemical Co. (St. Louis, MO). Ecolite scintillation fluid was obtained from ICN (Irvine, CA). All other reagent grade chemicals were obtained from Sigma Chemical Co. or Fisher Scientific (Pittsburgh, PA).

[³⁵S]GTPS autoradiography. Animals were sacrificed by decapitation, and the brains were removed and immersed in isopentane at 35°C. Twenty-micron coronal sections were cut on a cryostat and thaw-mounted onto gelatin-coated slides. Slides were rinsed in assay buffer (50 mM Tris-HCl, 3 mM MgCl₂, 0.2 mM EGTA, 100 mM NaCl, 0.1% (w/v) BSA, pH 7.4) at 25°C for 10 min. Slides were then incubated with 2 mM GDP in assay buffer at 25°C for 15 min. Sections were incubated with [³⁵S]GTPS (0.04 nM) and 2 mM GDP, with 10 µM WIN 55212-2 in assay buffer at 25°C for 2 hr. Basal [³⁵S]GTPS binding was assessed in the absence of agonist. Slides were rinsed twice in cold 50 mM Tris buffer and once in deionized water, dried and exposed to film for 48 hr. Films were digitized with a Sony XC-77 video camera and analyzed with the NIH Image program for Macintosh computers. Images were quantified by densitometric analysis with [¹⁴C] standards. Values are expressed as femtomoles of radioligand bound/mg tissue and corrected for [³⁵S] based on incorporation of [³⁵S] into brain paste standards (Sim et al., 1996b).

[³H]WIN 55212-2 receptor autoradiography. Brains were processed as described above and stored at 80°C until use. Slides were brought to room temperature and preincubated in assay buffer (20 mM HEPES with 0.5% (w/v) BSA and 1 mM MgCl₂) for 20 min at 30°C. Slides were incubated in 1 nM [³H]WIN 55212-2 in assay buffer for 80 min at 30°C. Nonspecific binding was assessed in the presence of 1 µM WIN 55212-2. Slides were rinsed four times for 10 min each in assay buffer at 25°C, then twice in deionized water at 4°C. Slides were dried thoroughly and exposed to Hyperfilm max for 3 weeks. Films were analyzed as described above. [³H] standards were used for quantification and values are expressed as femtomoles of radioligand bound/mg tissue.

Membrane preparations. Ten brain regions were dissected from fresh rat brains on ice. Tissue samples were pooled and homogenized with a Tissumizer (Tekmar, Cincinnati, OH) in cold membrane buffer (50 mM Tris-HCl pH 7.4, 3 mM MgCl₂, 1 mM EGTA) and centrifuged at 31,000 × g for 10 min at 4°C. Pellets were resuspended in membrane buffer, then centrifuged at 31,000 × g for 10 min at 4°C. Pellets were homogenized in membrane buffer, assayed for protein content (Bradford 1976) and stored in aliquots at 80°C until being assayed.

Agonist-stimulated [³⁵S]GTPS binding assays. Frozen membranes were thawed and diluted in membrane buffer, and centrifuged at 48,000 × g at 4°C for 10 min. Pellets were resuspended and homogenized in cold assay buffer (50 mM Tris-HCl pH 7.4, 3 mM MgCl₂, 0.2 mM EGTA, 100 mM NaCl), then assayed for protein (Bradford, 1976). For saturation binding analysis, membranes were incubated for 1 hr with 0.5 to 15 nM unlabeled GTPS in the presence or absence of 3 µM WIN 55212-2. WIN 55212-2 ED₅₀ and SR141716A K_e values were determined by incubating membranes for 2 hr with various concentrations of WIN 55212-2 (10-30,000 nM) in the presence and absence of 2 nM SR141716A. All assays included 10 to 20 µg of membrane protein and were conducted at 30°C with 0.1% (w/v) BSA, 30 µM GDP and 0.05 nM [³⁵S]GTPS in a final volume of 1 ml. Nonspecific binding was determined in the absence of WIN 55212-2 and the presence of 30 µM unlabeled GTPS. Reactions were terminated by rapid filtration under vacuum through Whatman GF/B glass fiber filters, followed by three washes with cold Tris buffer, pH 7.4. Bound radioactivity was determined by liquid scintillation spectrophotometry at 95% efficiency for [³⁵S] after overnight extraction of the filters in 4 ml Ecolite scintillation fluid.

[³H]SR141716A receptor binding assays. Membranes were prepared and incubated under the same conditions as for the [³⁵S]GTPS binding assays. Saturation binding analyses were performed by varying the concentration of [³H]SR141716A (0.02-2 nM) and incubating for 1 hr, and nonspecific binding was determined with 1 µM unlabeled SR141716A. IC₅₀ values for WIN 55212-2 were determined by varying the concentration of WIN 55212-2 (10- 30,000 nM) in the presence of 0.5 nM [³H]SR141716A and incubating for 2 hr. All binding assays included 3 to 10 µg of membrane protein and were conducted in [³⁵S]GTPS binding assay buffer (as described above), including 0.1% (w/v) BSA and 30 µM GDP. Assay tubes were incubated at 30°C and binding was terminated and bound radioactivity determined (at 45% efficiency for [³H]) as above.

[³H]WIN 55212-2 receptor binding assays. Frozen membranes were thawed and diluted in membrane buffer, and centrifuged at 48,000 × g at 4°C for 10 min. Pellets were resuspended and homogenized in 7 ml cold 20 mM HEPES-HCl, pH 8.0, with 1 mM MgCl₂, and assayed for protein (Bradford 1976). Saturation binding analyses were performed with 1 nM [³H]WIN 55212-2 plus 0.5 to 15 nM unlabeled WIN 55212-2 in a final volume of 1 ml including 0.1% (w/v) BSA. Nonspecific binding was determined in the presence of 4 µM WIN 55212-2. Assay tubes were incubated at 25°C for 90 min, and binding was terminated by rapid filtration under vacuum through Whatman GF/B glass fiber filters, followed by three washes with cold 20 mM HEPES-HCl, pH 8.0 buffer containing 1 mM MgCl₂ and 0.05% (w/v) BSA. Bound radioactivity was determined as above.

Data analysis. Net agonist-stimulated [³⁵S]GTPS binding values were calculated by subtracting basal binding values (absence of agonist) from agonist-stimulated values at each concentration of unlabeled GTPS. Binding analysis (including receptor binding, [³⁵S]GTPS saturation and agonist concentration-effect curves) was conducted by nonlinear regression with use of JMP for Macintosh (SAS, Cary, NC), or LIGAND (Munson and Rodbard, 1980). Mean amplification factors were calculated by dividing mean net agonist-stimulated [³⁵S]GTPS binding apparent B_max values by mean receptor binding B_max values for each receptor ligand. Standard error for each amplification factor value was calculated as the square root of the variance as estimated by the equation: where x1 and x2 are the mean B_max values for G-proteins and receptors, respectively, and s1 and s2 are the respective B_max standard errors. High-affinity fraction values (³H-labeled agonist/³H-labeled antagonist binding B_max ratios) and standard error for each region were calculated in the same manner. The K_e value for SR141716A was calculated by the equation: K_e = [Ant]/(dose-ratio  1) (Gaddum, 1957), where [Ant] is the concentration of SR141716A and dose-ratio is the ratio of WIN 55212-2 ED₅₀ values in the presence and absence of SR141716A, respectively. Significant differences in regional amplification factor and fraction of high-affinity agonist binding values were determined conservatively by performing multiple Student's t-tests to compare each region with every other at a significance level of  = .005 (Bonferoni adjustment to = .05 for 10 groups). Significant differences (P < .05) between other values were determined with JMP to perform Student's t-test for two groups, or analysis of variance and the Tukey-Kramer HSD test for multiple comparisons. Significant differences are indicated in the figures and graphs by letters: values that are not significantly different are denoted by the same single letter. Unless otherwise indicated, all data presented are the mean ± S.E.M. of three or more determinations from assays performed in triplicate.

	Results

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Regional differences in cannabinoid receptor binding and activated G-proteins by autoradiography. Previous autoradiographic studies have demonstrated that the distribution of cannabinoid-stimulated [³⁵S]GTPS binding in brain correlates with that of cannabinoid receptor binding (Herkenham et al., 1991b; Sim et al., 1995). For example, both receptor- and agonist-stimulated [³⁵S]GTPS autoradiography revealed the highest levels of receptor binding and activated G-proteins in the substantia nigra, entopeduncular nucleus and globus pallidus (Herkenham et al., 1991a; Sim et al., 1996a). To directly compare these two parameters, cannabinoid receptor ([³H]WIN 55212-2) and agonist-stimulated [³⁵S]GTPS binding were compared in the hippocampus, amygdala, thalamus, hypothalamus and cortex in coronal rat brain sections. Representative brain sections showing specific [³H]WIN 55212-2 binding and net WIN 55212-2-stimulated [³⁵S]GTPS binding are shown in coronal sections in figure 1. For both receptor binding and [³⁵S]GTPS autoradiography, the highest levels of binding were observed in the hippocampus and entopeduncular nucleus. However, visible differences between receptor and [³⁵S]GTPS binding could be observed in other regions. Intermediate levels of receptor binding were found in the thalamus, whereas the same area provided very low levels of agonist-stimulated [³⁵S]GTPS binding. In contrast, both amygdala and hypothalamus demonstrated intermediate-to-high levels of agonist-stimulated [³⁵S]GTPS binding, but only low levels of cannabinoid receptor binding. A particularly striking difference between cannabinoid receptor binding and activated G-proteins was found in the cortex, which showed an intermediate level of cannabinoid receptor binding, but high levels of agonist-stimulated [³⁵S]GTPS binding, particularly in the deeper layers of cortex. These results indicated that regional differences exist in the amplification of G-protein activity by cannabinoid receptors in the rat brain. However, it is important to note that these studies were conducted with only one concentration each of [³H]WIN 55212-2 or [³⁵S]GTPS, and cannot provide accurate determinations of the absolute ratio between receptors and agonist-activated G-proteins.

View larger version (62K): [in this window] [in a new window]   Fig. 1.   Representative autoradiograms of cannabinoid receptor binding and net cannabinoid-stimulated [35S]GTPS binding in coronal rat brain sections. Receptor binding was performed by incubating sections with 1 nM [3H]WIN 55212-2. Basal and cannabinoid-stimulated [35S]GTPS binding were performed by incubating sections with 0.04 nM [35S]GTPS and 2 mM GDP in the absence and presence of 10 µM WIN 55212-2, respectively. Both images were obtained by digital subtraction of autoradiograms: for receptor binding, nonspecific binding (with 1 µM unlabeled WIN 55212-2) was subtracted from total [3H]WIN 55212-2 binding; for [35S]GTPS binding, basal binding (determined in the absence of agonist) was subtracted from WIN 55212-2-stimulated binding.

Regional differences in cannabinoid receptor binding and activated G-proteins in membranes. When incubated in the presence of 30 µM GDP, WIN 55212-2 significantly increased the binding of [³⁵S]GTPS to rat brain membranes in a concentration-dependent and saturable manner in all regions examined: [³⁵S]GTPS binding was maximal in the presence of 3 µM WIN 55212-2 and was increased by 73 to 250% in all 10 regions, similar to previous results (Selley et al., 1996). Saturation binding analysis of the binding of [³⁵S]GTPS to cerebellar membranes in the presence and absence of WIN 55212-2 revealed that [³⁵S]GTPS bound with high (3 nM) and low (500-1000 nM) affinity. The agonist increased the number of high-affinity sites when added in the presence of micromolar concentrations of GDP (data not shown), analogous to the agonist effect observed in the mu and delta opioid systems (Breivogel et al., 1997). To isolate the effect of the agonist on the apparent K_D and B_max of [³⁵S]GTPS binding, basal binding was subtracted from agonist-stimulated binding at each concentration of GTPS. This method, which results in linear (monophasic) Scatchard plots, has been shown to yield values that correspond to the high-affinity binding of GTPS to receptor-activated G-proteins (Breivogel et al., 1997; Selley et al., 1997). To quantify differences in rat brain regional amplification factors, membrane saturation binding analyses of both cannabinoid receptors and cannabinoid receptor-activated G-proteins were conducted. Previous studies have shown that the number of G-proteins activated by a receptor can be determined by calculating the receptor/transducer amplification factor, i.e., the ratio between the B_max value for agonist-stimulated [³⁵S]GTPS binding and the B_max for receptor binding (Sim et al., 1996c). However, determination of B_max values for [³⁵S]GTPS binding is not simple because of the necessity for GDP and a lack of clear equilibrium conditions for the assay (see "Discussion"). For this reason, parameters are termed "apparent K_D" and "apparent B_max" for agonist-stimulated [³⁵S]GTPS binding.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Representative Scatchard plots of agonist ([3H]WIN 55212-2), antagonist ([3H]SR141716A) and net agonist-activated G-protein ([35S]GTPS) binding in two representative regions, frontal cortex (A) and thalamus (B). Scatchard analysis of all three radioligands was performed as described under "Materials and Methods." Net agonist-stimulated [35S]GTPS binding Scatchard plots were derived by subtracting basal from agonist-stimulated binding values at each concentration of unlabeled GTPS. In all cases, B/F values were obtained by dividing the concentration of bound ligand (in nM/mg of protein) by the concentration of free ligand (in nM). Data shown are representative of three or more experiments that showed similar results.

View this table: [in this window] [in a new window]   TABLE 2 Saturation binding analysis of cannabinoid receptor-stimulated [35S]GTPS binding in membranes from various rat brain regions Net WIN 55212-2-stimulated [35S]GTPS (cannabinoid receptor-activated G-protein) binding was determined as described under "Materials and Methods." The data represent mean values ± S.E.M. from saturation binding analyses which were conducted in triplicate three or more times in membranes from each region of rat brain. By the Tukey-Kramer HSD test at P < .05, values within a given column that are not significantly different are denoted by the same single letter. For the column of apparent KD values, none of the values are significantly different.

Receptor/transducer amplification factors and fractions of high-affinity agonist binding. Receptor/transducer amplification factors have been defined as the ratio of the apparent B_max of maximal receptor-stimulated [³⁵S]GTPS binding to receptor B_max, and reflects the relative number of G-proteins activated per receptor under receptor saturating conditions. Values for all regions are presented in figure 3. These ratios were calculated in two different ways. The total amplification factor (fig. 3A) was calculated from [³H]SR141716A binding B_max values and accounts for the total number of (coupled and uncoupled) receptor sites. The coupled amplification factor (fig. 3B) was calculated from high-affinity [³H]WIN 55212-2 receptor binding, and thus only considers high-affinity agonist binding (coupled) sites.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Receptor/G-protein amplification factors for each of 10 regions of rat brain. Panel A depicts the total amplification factor values, which are the ratios of net WIN 55212-2-stimulated G-proteins to [3H]SR141716A (antagonist)-binding cannabinoid receptors within each region. Panel B depicts coupled amplification factor values for each region, determined by high-affinity [3H]WIN 55212-2 binding to G-protein-coupled cannabinoid receptors. All data shown represent mean values ± S.E.M. for ratios calculated from at least three assays each. The shading of the bars indicates the significance grouping of the region as belonging to the low (open), moderate (shaded) or high (black) amplification factor groups. By Student's t-test, P < .005 for regions marked with different letters. Abbreviations for regions are as follows: Amyg, amygdala; B S, brainstem; Cblm, cerebellum; Coll, colliculi; Fr Ctx, frontal cortex; Hippo, hippocampus; Hypo, hypothalamus; S-M Ctx, sensomotor cortex; Thal, thalamus.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Fractions of high-affinity agonist binding to cannabinoid receptors in 10 regions of rat brain. Fractions of high-affinity binding were calculated as the ratio of [3H]WIN 55212-2 Bmax (coupled receptors) to [3H]SR141716A Bmax (total receptors). Data shown are mean values ± S.E.M. for ratios that were calculated from three or more experiments each. *P < .05 that [3H]SR141716A Bmax is significantly different from [3H]WIN 55212-2 Bmax by Student's t-test. Abbreviations for regions are as indicated in figure 3.

Relationship of receptor/transducer amplification factors to receptor occupancy. The previous determinations of amplification factors were made with a concentration of the agonist WIN 55212-2 (3 µM) that resulted in near-complete receptor occupancy and maximal stimulation of [³⁵S]GTPS binding. To determine whether receptor/transducer amplification factors were different at lower levels of receptor occupancy, three representative regions were assayed with various concentrations of WIN 55212-2 in the presence and absence of 2 nM SR141716A, and for receptor binding with the same concentrations of WIN 55212-2 under [³⁵S]GTPS binding conditions (with sodium and GDP). Because ³H-labeled agonist binding cannot practically be conducted under [³⁵S]GTPS binding conditions, WIN 55212-2 displacement of [³H]SR141716A was measured. The cerebellum, amygdala and hypothalamus were chosen because they represent the full range of measured receptor densities and amplification factors (see table 1 and fig. 3). The cerebellum is among the highest regions for receptor density, but lowest for amplification; the hypothalamus exhibits low receptor density and the highest amplification; and the amygdala has intermediate values.

View this table: [in this window] [in a new window]   TABLE 3 Potencies of WIN 55212-2 in stimulating [35S]GTPS and displacing [3H]SR141716A binding ED50 values and IC50 values were determined for WIN 55212-2 stimulation of [35S]GTPS binding and WIN 55212-2 displacement of [3H]SR141716A binding simultaneously by incubating membranes with either 0.05 nM [35S]GTPS or 0.05 nM unlabeled GTPS (for [3H]SR141716A), along with 30 µM GDP and 30 to 10,000 nM WIN 55212-2. Ki values were calculated by the equation: Ki = IC50/([L]/KD) + 1) (Cheng and Prusoff, 1973), where [L] was the concentration of [3H]SR141716A in each assay and KD values were those for [3H]SR141716A shown in table 1. Ki/ED50 ratios were calculated for each assay of each region to obtain mean ± S.E.M. values from three to five determinations. Values that are not significantly different (by Tukey-Kramer test at P < .05) are denoted with the same single letter.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Relationship of G-protein activation to receptor occupancy by WIN 55212-2. (A) Effect of varying concentrations of WIN 55212-2 on activation of G-proteins ([35S]GTPS) and receptor occupancy (displacement of [3H]SR141716A binding) in cerebellar membranes with [35S]GTPS binding conditions. Data are calculated as picomoles of G-proteins activated per milligram of protein, or picomoles of WIN 55212-2 bound per milligram of protein, respectively, as described in table 3. (B) The receptor/G-protein amplification factors determined by dividing the number of activated G-proteins by the number of agonist-occupied receptors for each concentration of WIN 55212-2 in each assay. Data are mean values ± S.E.M. from three or more experiments, each performed in triplicate.

	Discussion

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The present study used agonist-stimulated [³⁵S]GTPS autoradiography (Sim et al., 1995) and membrane saturation binding analysis (Gierschik et al., 1991; Traynor and Nahorski, 1995; Sim et al., 1996c) to compare levels of G-protein activation by cannabinoid receptors in different regions of the rat brain. In the autoradiographic analysis, relative levels of G-protein activation and receptor binding were compared on a qualitative basis and provided the rationale for conducting the saturation binding analyses in brain membranes. The basis of this reaction is an agonist-induced increase in the number of binding sites which exhibited high affinity for [³⁵S]GTPS. Thus, the decrease in the apparent K_D of [³⁵S]GTPS binding (from 500-1000 nM to the values in table 2) represents the agonist-induced increase in the probability that a G-protein will bind [³⁵S]GTPS (or GTP) and become activated. However, it is clear that the [³⁵S]GTPS binding assay is more complex than a simple increase in [³⁵S]GTPS affinity. It is also apparent that cannabinoid agonists also decrease the IC₅₀ of GDP in inhibiting [³⁵S]GTPS binding, i.e., the agonist lowers the affinity of the activated G-protein for GDP (C.S. Breivogel and S.R. Childers, unpublished observations), a finding we previously reported in the opioid system (Selley et al., 1997). Nevertheless, from a practical point of view, the increase in [³⁵S]GTPS binding that occurs as a result of the increase in [³⁵S]GTPS affinity provides the basis for detecting receptor/G-protein coupling.

Another potential issue in analyzing these data is the question of equilibrium for [³⁵S]GTPS binding. Previous studies have suggested that [³⁵S]GTPS binding to G-proteins is "quasi-irreversible" (Pfeuffer and Helmreich, 1975). However, in brain membranes with cannabinoid-stimulated [³⁵S]GTPS binding, this does not appear to be the case. Kinetic experiments in cerebellar membranes revealed that cannabinoid-stimulated [³⁵S]GTPS binding associated with a t_1/2 of approximately 40 min and dissociated (using excess unlabeled GTPS) with a t_1/2 of 30 min in the presence of 3 µM WIN 55212-2 and 30 µM GDP (C. S. Breivogel and S. R. Childers, unpublished observations). Thus, although the equilibrium of [³⁵S]GTPS binding depends on many factors including agonist-receptor, receptor-G-protein, G-, G-GDP and G-[³⁵S]GTPS interactions, it may be subject to equilibrium binding data analysis if conducted under appropriate and defined assay conditions. It should be noted that absolute binding parameters for [³⁵S]GTPS binding depends on the concentration of GDP. Because 30 µM GDP was used in these studies, the terms "apparent K_D" and "apparent B_max" refer to saturation binding parameters obtained in [³⁵S]GTPS binding assays.

The ratio of apparent B_max values from WIN 55212-2-stimulated [³⁵S]GTPS binding and B_max values of cannabinoid receptor binding yields receptor/G-protein amplification factors, which can be interpreted as a relative measure of the number of G-proteins that a cannabinoid receptor is able to activate. It should be noted that these amplification factors are not absolute and may be higher under physiological conditions in intact systems. The [³⁵S]GTPS binding assay may underestimate physiological amplification for two reasons. First, there are differences in the binding of GTPS, a slowly hydrolyzable GTP analog, compared with GTP. In intact cells, GTP is the substrate for the activated G-protein, which hydrolyzes the GTP to GDP to become inactivated. Because [³⁵S]GTPS is so poorly hydrolyzed, the G-protein is unable to cycle and become activated more than once. Even if the G-protein were able to cycle, this technique measures only the [³⁵S]GTPS bound at the termination of the assay. Second, it is not clear what proportion of the ligand-binding receptors are involved in the activation of G-proteins. However, it appears that maximal WIN 55212-2-stimulated [³⁵S]GTPS binding requires full occupancy of membrane cannabinoid receptors, because the G-protein activation and receptor occupancy curves for WIN 55212-2 are parallel and exhibit similar ED₅₀ and K_i values, respectively.

With either autoradiography or membrane saturation binding analysis, there were many significant differences in regional amplification factor values. From autoradiographic analysis, the relatively few cannabinoid receptors in the amygdala and hypothalamus appeared to activate nearly as many G-proteins as in the receptor-dense hippocampus or entopeduncular nucleus, and more G-proteins than in the thalamus or cortex, which had intermediate receptor densities. Membrane assay saturation binding analyses yielded total (³H-labeled antagonist) amplification factor values that varied over nearly a 4-fold range from 2.0 to 7.5 G-proteins activated per receptor. Coupled (³H-labeled agonist) amplification factor values varied over a 3-fold range from 2.3 to 6.8 G-proteins per receptor. Moreover, the fractions of high-affinity agonist binding in 2 of the 10 regions assayed were significantly less than one, and thus had a significant fraction of receptors that remained uncoupled from G-proteins under assay conditions that favored such coupling.

Although there were many significant differences in amplification factors among different brain regions, the values for cannabinoid receptors were all substantially lower than those previously calculated for mu and delta opioid receptors in rat striatum. In the previous study, striatal opioid receptors activated approximately 20 G-proteins per agonist-binding receptor (Sim et al., 1996c), whereas in both the previous and present studies agonist-binding cannabinoid receptors activated between 2 and 7.5 G-proteins. This demonstrates that cannabinoid receptors in brain couple with relatively low efficiency to G-proteins when compared with opioid receptors.

In previous studies of the cannabinoid receptor system (Sim et al., 1996c), amplification factors were calculated by agonist binding to determine receptor numbers. High-affinity binding of [³H]WIN 55212-2 is sensitive to guanine nucleotides, Na⁺ and Mg⁺⁺, which indicates that it is dependent on coupling of cannabinoid receptors to G-proteins (Devane et al., 1988), and therefore only measures those receptors that are coupled to a G-protein under the conditions of the agonist binding assay. Such an analysis will usually yield a lower B_max (and thus a higher amplification factor value) than when receptor binding is performed with an antagonist. The current study used both an agonist and an antagonist to determine receptor levels in the various brain regions. In some regions, determination of receptor density by high-affinity agonist binding was complicated by the appearance of both high (3 nM) and low (>100 nM) affinity [³H]WIN 55212-2 binding sites. The low-affinity agonist sites were most likely uncoupled cannabinoid receptors, because the unlabeled antagonist displaced a high concentration of [³H]WIN 55212-2 (12 nM) with high affinity, and the cannabinoid agonist CP55940 displaced the same concentrations of [³H]WIN 55212-2 with low affinity (data not shown). These findings were consistent with the coupled and uncoupled agonist affinity states traditionally observed with G-protein-coupled receptors. Although [³H]WIN 55212-2 binding analysis was conducted on both one- and two-site fits by LIGAND, this low-affinity site was responsible for a higher degree of variability in calculated high-affinity agonist binding values of some regions. For example the presence of a relatively large proportion of low-affinity sites was probably the reason that the K_D of [³H]WIN 55212-2 in the colliculi was twice that of any other region. In contrast, the binding of the antagonist, [³H]SR141716A, is not sensitive to either guanine nucleotides or sodium (Rinaldi-Carmona et al., 1996), and only one site was detected in all regions, even in the presence of 100 mM NaCl and 30 µM GDP. Antagonist binding therefore measures receptor B_max values more reliably than agonist binding. Furthermore, antagonist receptor binding has the advantage of being more readily comparable with, and more relevant to, agonist-stimulated [³⁵S]GTPS binding. The presence of GDP and sodium in both the [³⁵S]GTPS and [³H]SR141716A binding assays induces cannabinoid receptors to a low-affinity state for agonist (WIN 55212-2). In contrast, ³H-labeled agonist binding is performed in the absence of GDP and sodium to provide high-affinity agonist binding to cannabinoid receptors, which is not present under [³⁵S]GTPS binding conditions. Because the concentration of WIN 55212-2 used to stimulate [³⁵S]GTPS binding is sufficient to saturate all high- and low-affinity cannabinoid receptors, ³H-labeled antagonist measurements of receptor numbers are more relevant to the activation of G-proteins measured in the [³⁵S]GTPS binding assay.

The finding of differences in amplification factors across regions was observed in both membranes and by in vitro autoradiography of brain sections. However, some quantitative differences were also observed which were probably caused by the differences in anatomical resolution of the two methods. Small nuclei with high levels of cannabinoid receptors were differentially included in dissected brain regions. For example, the thalamus membrane preparation contained tissue from the entopeduncular nucleus, the brainstem contained substantia nigra and the striatum included globus pallidus. Moreover, such dissections inevitably lose the spatial arrangement (e.g., rostral-caudal differences) that are clearly demonstrated by autoradiography. Thus, although membrane saturation binding analysis has the advantage of being more quantifiable, the fine anatomical resolution of autoradiographic analysis was unattainable by regional dissection.

As defined in this study, amplification factors were calculated at saturating concentrations of agonist. However, in brain it is difficult to predict how often cannabinoid receptors will be saturated with agonist. For this reason, it was of interest to calculate amplification factors at varying receptor occupancy. Figure 5 shows that there was little effect of agonist concentration (i.e., receptor occupancy) on the catalytic amplification factors between 100 and 10,000 nM WIN 55212-2. The amplification factor was difficult to calculate precisely at low agonist concentrations (<100 nM) because of the low values for both activated G-proteins and occupied receptors. Nevertheless, there was a significant increase in amplification factor observed in cerebellum at the lowest concentration of WIN 55212-2 used, 30 nM. It was interesting that in the three regions examined for K_i/ED₅₀ ratios, only the cannabinoid receptor-rich cerebellum had a ratio greater than one (implying a slight receptor reserve), whereas the intermediate and low receptor density regions, amygdala and hypothalamus, had K_i/ED₅₀ ratios equal to one. The slight receptor reserve in cerebellum predicts the increase in amplification factor at low concentrations of WIN 55212-2 (fig. 5B): because of the existence of receptor reserve, low receptor occupancy results in a relatively higher level of transducer activation. Furthermore, as the activation of transducer becomes maximal and levels off with increasing agonist concentration, receptor occupancy continues to increase which results in a decreasing ratio of activated G-proteins to occupied receptors. If any G-protein-coupled receptor system in rat brain were to exhibit reserve for the activation of G-proteins, it might be the cannabinoid system, because there is a large excess of cannabinoid receptors compared with other known G-protein-coupled receptors (Herkenham et al., 1991b; Sim et al., 1996c). The fact that there was only slight receptor reserve observed in cerebellum, and none in the other regions, is probably because of the great excess of G-proteins in rat brain (Asano et al., 1990) even when compared with the number of CB1 receptors (table 1). The fact that some reserve was measured in a region with high receptor density agrees well with results in the mu opioid system: K_i/ED₅₀ ratios were higher in a mu receptor-transfected cell line with high receptor density (approximately equal to cerebellar CB1 density) than in brain membranes with lower mu receptor density (D. E. Selley and S. R. Childers, unpublished observations).

The mechanisms underlying the differences in receptor/G-protein amplification factors are not yet clear, and will be the focus of future studies. One possibility, as discussed above, is that cannabinoid receptor subtypes (e.g., the splice variants, CB1 and CB1A) may exhibit different levels of catalytic activity. Thus, the regional variation in amplification factors may reflect differences in the ratio of these (and perhaps yet undiscovered) subtypes. Alternatively, the regional differences in amplification factors may in part be caused by the co-localization of the different subtypes of G-protein  subunits with cannabinoid receptors. Subtypes of G_i/o  subunits (G_i1, G_i2, G_i3, G_o1 and G_o2) (Jones and Reed, 1987; Hsu et al., 1990) may be activated specifically or with varying degrees of efficiency by cannabinoid receptors, as seen in other receptor systems (McKenzie and Milligan, 1990; Senogles et al., 1990). If CB1 receptors activate different G-protein  subunits with varying efficiency, then agonist concentration might affect the composition of the activated subtypes. For instance, low receptor occupancy could preferentially activate one subtype, and additional subtype(s) could be recruited at higher concentrations of agonist. Similarly, G-protein subunits have been shown to confer receptor specificity or selectivity of G-protein coupling even when paired with the same  subunit subtype (Kleuss et al., 1992). Thus, regional amplification factors could vary with the composition of G-protein subunit subtypes present in each region. However, the similarity of the regional apparent K_D values for agonist-stimulated [³⁵S]GTPS binding indicate that cannabinoid receptors are activating the same class of G-protein  subunits (G_i and G_o as opposed to G_s or other subtypes) because, for example, G_s binds GTP analogs with much greater affinity than G_i or G_o (Rasenick and Childers, 1989). This also agrees with previous reports of cannabinoids acting via pertussis toxin-sensitive (G_i/o-mediated) mechanisms (Childers and Deadwyler, 1996).

Another factor that might contribute to the regional variation of amplification factors is the ratio of receptors to G-proteins. This model is represented by the law of mass action: the higher the concentration of available G-proteins, the more frequently a receptor and G-protein will collide in an interaction that results in the activation of the G-protein. The observation that the highest amplification factors occur in the lower receptor density regions, and that the high-density regions all have low amplification factors supports this hypothesis.

Another explanation is that amplification factors would depend on the degree of cannabinoid receptor/G-protein precoupling. That possibility was investigated in the present study by correlating amplification factors with the fractions of high-affinity agonist binding. These data (correlated from figs. 3 and 4) showed that the fraction of high-affinity binding correlated poorly with both coupled amplification factors (r = 0.3) and total amplification factors (r = 0.6) (data not shown). Therefore, the fraction of high-affinity binding is not an important determinant of amplification factor values.

Regional differences in receptor/G-protein amplification factors may help to elucidate the physiological significance of the endogenous cannabinoid system. For example, the hippocampus has high levels of both cannabinoid-activated G-proteins and cannabinoid receptors. Thus, the effects of cannabinoids on short-term memory, which appear to be mediated by the hippocampus (Deadwyler et al., 1995), might be predicted based on the high receptor density. This is in contrast to the hypothalamus, a region with a high level of cannabinoid-activated G-proteins despite low cannabinoid receptor density. The well-established effects of cannabinoids on basal body temperature and hypothalamic hormone function (Dewey 1986; Hollister 1986) would not necessarily be predicted based on the relatively low density of cannabinoid receptors in the hypothalamus. Thus, in this region, receptor activation of G-proteins may be a better predictor of cannabinoid efficacy than cannabinoid receptor levels. Therefore, predictions of the magnitude of a drug effect in a given brain region must be made not only on the basis of receptor binding analysis, but also on the degree of activation of intracellular signal transduction mechanisms by those receptors.