Introduction

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Agonist efficacy is defined as the maximal effect of a drug in the production of a particular biological response. However, it is well-established that the efficacy of a particular drug at a specific receptor type is not always constant among different responses and different tissues, partly because of variations in receptor density and receptor reserve. Receptor reserve occurs when a "full" agonist produces a maximal response at less than full receptor occupancy (Stephenson, 1956; Furchgott, 1966). Thus, a functional definition of intrinsic efficacy must reflect both the maximal effect produced and the level of receptor occupancy required to produce that effect by a particular agonist within a given system (Kenakin, 1993).

Opiates and synthetic opioid drugs are widely recognized as effective analgesics that bind with high affinity to mu type (OP-3) opioid receptors (Corbett et al., 1993; Dhawan et al., 1996). Mu opioid receptors are members of the superfamily of G-protein-coupled receptors (Chen et al., 1993; Thompson et al., 1993; Wang et al., 1994); they are coupled through G-proteins of the pertussis toxin-sensitive G_i/G_o family (Fedynyshyn and Lee, 1989a; Selley and Bidlack, 1992; Laugwitz et al., 1993; Chakrabarti et al., 1995) to effectors including inhibition of adenylyl cyclase (Yu and Sadee, 1988; Fedynyshyn and Lee, 1989b; Childers, 1991), stimulation of potassium channel conductance (Aghajanian and Wang, 1986; North et al., 1987; Chen and Yu, 1994) and inhibition of calcium channel conductance (Moises et al., 1994; Rhim and Miller, 1994). Initiation of G-protein activation by receptors decreases the affinity of the  subunit for GDP relative to GTP, thus promoting guanine nucleotide exchange (Gilman, 1987; Florio and Sternweiss, 1989). The receptor activates G-proteins catalytically, and each receptor can activate multiple G-proteins (Asano et al., 1984; Gierschik et al., 1991; Sim et al., 1996b). This initial stage of G-protein activation can be measured in isolated membranes by assaying agonist-stimulated binding of the hydrolysis-resistant GTP analog, [³⁵S]GTPS, in the presence of excess GDP (Hilf et al., 1989; Lorenzen et al., 1993; Sim et al., 1995; Traynor and Nahorski, 1995). Recent studies have determined that agonist efficacy for G-protein activation can be measured as maximal stimulation of [³⁵S]GTPS binding (Traynor and Nahorski, 1995; Emmerson et al., 1996; Lorenzen et al., 1996; Selley et al., 1997)_.

Saturation analysis of agonist-stimulated [³⁵S]GTPS binding can be used to determine both the apparent affinity of [³⁵S]GTPS for the activated G-protein, and the number of G-proteins activated, under the particular assay conditions used (Gierschik et al., 1991; Tian et al., 1994; Sim et al., 1996b; Selley et al., 1997). Recent studies in our laboratory showed that agonist efficacy for G-protein activation by mu opioid receptors was related to both the apparent affinity of the G-protein for [³⁵S]GTPS in the presence of GDP and the apparent number of G-proteins activated by the agonist-occupied receptor (Selley et al., 1997). Moreover, differences in efficacy among several mu opioid agonists also were magnified by increasing GDP concentrations and by decreasing receptor density. Thus, the mechanisms underlying agonist efficacy for G-protein activation by agonists acting at mu opioid receptors appear to be complex. Intrinsic properties of the agonists that determine the level of receptor activation as well as system-dependent factors, such as receptor density and the equilibrium between G-protein activation and inactivation, are all important determinants of the agonist-induced response.

This study further examined the relationship between intrinsic agonist efficacy and the underlying mechanisms of G-protein activation by agonist-occupied mu opioid receptors in membranes from CHO cells transfected with cDNA encoding the mouse mu receptor (mMOR-CHO cells) (Abood et al., 1995; Kaufman et al., 1995) and from rat thalamus, a brain region enriched in mu opioid receptors (Herkenham and Pert, 1982; Sim et al., 1995, 1996a). With use of agonist-stimulated [³⁵S]GTPS binding as a direct measurement of G-protein activation, efficacy is defined as the maximal stimulation produced by an agonist, and intrinsic efficacy is defined as a combination of the maximal stimulation and the level of receptor occupancy at which half-maximal stimulation is produced by an agonist (K_i/EC₅₀ ratio). These parameters are combined according to the relationship introduced by Ehlert (1985) to quantify the effects of muscarinic cholinergic agonists in stimulating adenylyl cyclase activity, and which recently has been used to describe the intrinsic efficacies of agonists for stimulation of [³⁵S]GTPS binding in delta opioid receptor-transfected CHO cells (Quock et al., 1997).

	Materials and Methods

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Materials. [³⁵S]GTPS (1150-1300 Ci/mmol) was purchased from New England Nuclear Corp. (Boston, MA). mMOR-CHO cells were generously provided by Drs. Lawrence Toll and Christopher Evans. Ecolite scintillation fluid was obtained from ICN (Irvine, CA). DAMGO, naloxone and nalbuphine were purchased from Sigma Chemical Co. (St. Louis, MO). Geneticin (G-418) and penicillin-streptomycin were purchased from Gibco/BRL (Grand Island, NY). All other nonpeptide opioid agonists were obtained from the NIDA drug supply program (Research Triangle Institute, Research Triangle Park, NC). FBS and Geneticin (G-418) were purchased from Gibco/BRL. Guanosine-5'-O-(-thio)-triphosphate and guanosine-5'-diphosphate were purchased from Boehringer Mannheim (New York, NY). All other chemicals (reagent grade) were obtained from Sigma Chemical Co. or from Fisher Scientific Co. (Pittsburgh, PA).

Cell culture. Cells were cultured at 37°C in a humidified atmosphere of 5% CO₂ and 95% air in 50% DMEM and 50% F-12 Nutrient Mixture (Ham) containing 100 units/ml penicillin, 100 µg/ml streptomycin and 5% FBS. Cells were harvested by replacing the media with cold phosphate-buffered saline containing 0.04% ethylenediaminetetraacetic acid for 5 min, followed by agitation, and collected by centrifugation at 345 × g for 10 min.

Membrane preparation. Rats were sacrificed by decapitation and the thalamus was dissected on ice. mMOR-CHO cells or rat thalami were homogenized in 20 vol ice-cold 50 mM Tris-HCl, 3 mM MgCl₂, 1 mM EGTA, pH 7.4 (membrane buffer) with a Polytron. The homogenate was centrifuged at 48,000 × g at 4°C for 10 min, resuspended in membrane buffer, centrifuged again at 48,000 × g at 4°C for 10 min and finally resuspended in 50 mM Tris-HCl, 3 mM MgCl₂, 0.2 mM EGTA, 100 mM NaCl, pH 7.4 (assay buffer). Membrane protein levels were determined by the method of Bradford (1976).

[³⁵S]GTPS binding assays. Agonist-stimulated [³⁵S]GTPS binding was assayed as described previously (Selley et al., 1997). For concentration-effect curves, mMOR-CHO cell (25 µg protein) or rat thalamic (10 µg protein) membranes were incubated for 1 hr at 30°C, with and without various drugs, in assay buffer containing 0.05 nM [³⁵S]GTPS and 10 µM (mMOR-CHO) or 30 µM (rat thalamus) GDP. Basal binding was assessed in the presence of GDP and absence of drug. Nonspecific binding was measured in the presence of 10 µM GTPS. For Scatchard analysis, mMOR-CHO cell membranes were incubated with 0.1 nM [³⁵S]GTPS and 0.1 to 30 nM unlabeled GTPS in the presence of 10 µM GDP, with and without various drugs, in assay buffer for 1 hr at 30°C. Scatchard analysis of [³⁵S]GTPS binding in rat thalamic membranes was conducted similarly with 30 µM GDP and 0.5 to 50 nM unlabeled GTPS, and was incubated for 2 hr at 30°C. The incubation was terminated by rapid filtration under vacuum through Whatman GF/B glass fiber filters, followed by three washes with 3 ml ice-cold 50 mM Tris-HCl, pH 7.4 (Tris buffer). Bound radioactivity was determined by liquid scintillation spectrophotometry at 95% efficiency for ³⁵S after overnight extraction of the filters in 5 ml Ecolite scintillation fluid.

Receptor binding assays. Membranes, prepared from mMOR-CHO cells (50 µg protein) or rat thalamus (125-150 µg protein), were incubated for 1 hr at 30°C with 1 nM [³H]naloxone in assay buffer containing 10 µM (mMOR-CHO) or 30 µM (rat thalamus) GDP, 0.05 nM GTPS and 0 to 100 nM unlabeled naloxone or 0 to 30 µM unlabeled opioid agonists. Nonspecific binding was determined with 10 µM unlabeled naloxone. Reactions were terminated by rapid filtration through glass fiber filters, and the filters were rinsed three times with ice-cold Tris buffer. Bound radioactivity was determined by liquid scintillation spectrophotometry at 50% efficiency for ³H after overnight extraction of the filters in 5 ml Ecolite scintillation fluid.

Data analysis. Unless otherwise indicated, data are reported as mean values ± S.E. of at least three separate experiments, each of which were performed in triplicate. Net-stimulated [³⁵S]GTPS binding is defined as stimulated binding minus basal binding. Percent stimulation is defined as (net stimulated binding/basal binding) × 100%; %E_max is defined as the maximum percent stimulation by an agonist, as determined by nonlinear regression analysis of concentration-effect curves. Percent maximal stimulation is defined as (net stimulated binding by agonist/net stimulated binding by 10 µM DAMGO) × 100%; %_Max is defined as the maximum stimulation derived from the concentration-effect curve of the percent maximal stimulation by an agonist, as determined by nonlinear regression analysis. These parameters, which were determined within each individual experiment by the inclusion of 10 µM DAMGO, as well as EC₅₀ values, were calculated by nonlinear regression analysis of concentration-effect curves with JMP (SAS Institute, Cary, NC) with an iterative model. Correlation analyses were performed by linear regression with JMP. Statistical significance of the data was determined by analysis of variance, followed by the nonpaired two-tailed Student's t-test with JMP. Saturation analyses were conducted by Scatchard plots with use of EBDA and LIGAND (Munson and Rodbard, 1980). Because the presence of GDP precludes determination of absolute K_D and B_max values from [³⁵S]GTPS saturation analysis, these values are termed "apparent" [³⁵S]GTPS K_D and B_max values. K_i values were determined by the Cheng-Prusoff relationship (Cheng and Prusoff, 1973). K_i/EC₅₀ ratios were determined to be significantly different from one when the two values (K_i and EC₅₀) were significantly different from each other. Intrinsic efficacy values were determined according to a modification of the relationship described previously by Ehlert (1985):

(1)

For partial agonists, the K_i/EC₅₀ ratio is assumed to be one, and the expression reduces to:

(2)

	Results

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Relationship between agonist-stimulated [³⁵S]GTPS binding and receptor binding. To determine the intrinsic efficacy of opioid agonists for G-protein activation, relative maximal stimulation (%_Max), EC₅₀ and K_i/EC₅₀ ratios were measured in concentration-effect curves for agonist-stimulated [³⁵S]GTPS binding and displacement of [³H]naloxone binding, under the same assay conditions, in mMOR-CHO cell and rat thalamic membranes. Results in mMOR-CHO membranes are shown in figure 1A and table 1A. Because DAMGO previously was found to produce the greatest stimulation of [³⁵S]GTPS binding among several opioid agonists (Selley et al., 1997), stimulation by each agonist is presented as a percentage of the maximal stimulation produced by DAMGO in each experiment. Nonlinear regression analysis of concentration-effect curves revealed that the maximum absolute percent stimulation (%E_max) of [³⁵S]GTPS binding by DAMGO was 523 ± 23% over basal in mMOR-CHO membranes. DAMGO, methadone, sufentanil and alfentanil were all full agonists in mMOR-CHO membranes, as observed previously with DAMGO, morphine and fentanyl (table 1A) (Selley et al., 1997). However, meperidine was a partial agonist of moderate efficacy, similar to buprenorphine (table 1A) (Selley et al., 1997). Nalorphine and nalbuphine produced the least stimulation, similar to levallorphan (table 1A) (Selley et al., 1997).

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Concentration-effect relationship of opioid stimulation of [35S]GTPS binding and competition for [3H]naloxone binding in mMOR-CHO cell membranes. Membranes were incubated with 10 µM GDP, various concentrations of opioid agonists, and (A) 0.05 nM [35S]GTPS or (B) 1 nM [3H]naloxone with 0.05 nM unlabeled GTPS. Data are mean ± S.E. of: (A) percent of maximal stimulation produced by 10 µM DAMGO or (B) percent of [3H]naloxone bound in the absence of competing ligand. Basal [35S]GTPS binding was 23.9 ± 1.5 fmol/mg protein. Control [3H]naloxone binding was 1.27 ± 0.07 pmol/mg protein. The EC50, %Max and Ki values from curve-fitting of these data are shown in table 1A.

View this table: [in this window] [in a new window]   TABLE 1 [35S]GTPS intrinsic efficacies and [3H]naloxone Ki values of opioid agonists in mMOR-CHO cell and rat thalamic membranes Data were obtained from experiments depicted in figures 1 and 2. For calculations of intrinsic efficacy, Ki/EC50 values for partial agonists are assumed to be 1; where Ki/EC50 values are 1, intrinsic efficacy was determined by the fractional Emax. Emax values containing the same letter designations are not significantly different from each other, whereas those that do not contain any similar letter designations are significantly different (P < .05). For Ki/EC50 ratios: *P < .05; **P < .01 different from 1, respectively.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Concentration-effect relationship of opioid stimulation of [35S]GTPS binding and competition for [3H]naloxone binding in rat thalamic membranes. Membranes were incubated with 30 µM GDP, various concentrations of opioid agonists, and (A) 0.05 nM [35S]GTPS or (B) 1 nM [3H]naloxone with 0.05 nM unlabeled GTPS. Data are mean ± S.E. of: (A) percent of maximal stimulation produced by 10 µM DAMGO or (B) percent of [3H]naloxone bound in the absence of competing ligand. Basal [35S]GTPS binding was 72.3 ± 7.8 fmol/mg protein. Control [3H]naloxone binding was 0.22 ± 0.01 pmol/mg protein. The EC50, %Max and Ki values from curve-fitting of these data are shown in table 1B.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Scatchard plots of [3H]naloxone binding to mMOR-CHO cell and rat thalamic membranes. Membranes were incubated with 1 nM [3H]naloxone, 10 µM (mMOR-CHO) or 30 µM (rat thalamus) GDP and 0.05 nM unlabeled GTPS in the presence and absence of 0.2 to 100 nM unlabeled naloxone. Data shown are representative experiments that were each replicated three to four times with similar results.

Saturation analysis of agonist-stimulated [³⁵S]GTPS binding. Saturation analysis of net agonist-stimulated [³⁵S]GTPS binding to mMOR-CHO cell membranes (fig. 4) was conducted with agonists of different efficacies to examine differences in the apparent affinity of agonist-induced GTPS binding to G-proteins (K_D of net-stimulated [³⁵S]GTPS binding) and in the apparent number of G-proteins activated by agonist-occupied receptors (B_max of net-stimulated [³⁵S]GTPS binding), as described previously (Selley et al., 1997). Results (table 2) showed that all full agonists in mMOR-CHO cell membranes produced similar K_D values (1-1.5 nM) for net-stimulated [³⁵S]GTPS binding, whereas all partial agonists produced apparent K_D values significantly greater than those obtained with full agonists. There also appeared to be a general trend toward an increase in the apparent B_max of [³⁵S]GTPS binding produced by full agonists compared with partial agonists, although there was greater variability in this parameter than in the K_D values. In particular, significant differences in B_max values were obtained between full- and low-efficacy partial agonists (e.g., DAMGO versus nalbuphine), but fewer significant differences were obtained among drugs that were full agonists in mMOR-CHO membranes (but partial agonists in thalamic membranes) and intermediate efficacy partial agonists (e.g., sufentanil versus meperidine).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Homologous displacement of basal and agonist-stimulated [35S]GTPS binding in mMOR-CHO cell membranes. Membranes were incubated with 0.1 nM [35S]GTPS, 10 µM GDP and 0.1 to 30 nM unlabeled GTPS in the presence and absence of maximally effective concentrations of each respective agonist. Data shown are the mean percent of control [35S]GTPS binding (binding measured in the absence of agonist or unlabeled GTPS) ± S.E. Control [35S]GTPS binding was 61.1 ± 1.4 fmol/mg protein. The KD and Bmax values from saturation analysis of these data are given in table 2.

View this table: [in this window] [in a new window]   TABLE 2 KD and Bmax values for net agonist-stimulated [35S]GTPS binding in mMOR-CHO cell and rat thalamic membranes Data were obtained from experiments depicted in figure 4. Values containing the same letter designations are not significantly different from each other, whereas those that do not contain any similar letter designations are significantly different (P < .05).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Correlation of agonist intrinsic efficacy values with the corresponding Bmax/KD values of agonist-stimulated [35S]GTPS binding. Agonist intrinsic efficacy for stimulation of 0.05 nM [35S]GTPS binding was correlated with the Bmax/KD of agonist-stimulated [35S]GTPS binding determined by saturation analysis of [35S]GTPS binding in the presence of maximally effective concentrations of each agonist in (A) mMOR-CHO cell membranes and (B) rat thalamic membranes. Intrinsic efficacy values were obtained from table 1, and Bmax/KD values were calculated from the data in table 2. Abbreviations: Mtd, methadone; DMG, DAMGO; Fnt, fentanyl; Mrp, morphine; Alf, alfentanil; Suf, sufentanil; Mep, meperidine; Bpr, buprenorphine; Nlr, nalorphine; Nlb, nalbuphine; Lev, levallorphan.

	Discussion

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The present study revealed several important findings that are relevant to the concepts of agonist efficacy and receptor reserve at G-protein-coupled receptors. First, the relationship between receptor occupancy and G-protein activation by mu opioid receptors depended on receptor density, as predicted from classical receptor theory (Furchgott, 1966; Kenakin, 1993). Second, the absolute magnitude of the stimulation was higher in the system with the higher receptor density. This also was expected because increases in the magnitude of agonist-stimulated responses with increasing receptor density has been reported in cells transfected with other G-protein-coupled receptors (Boddeke et al., 1992; Varrault et al., 1992; Prather et al., 1994; MacEwan et al., 1995). However, it should be noted that increases in maximal response as a function of receptor density at one level of the signal transduction pathway do not necessarily result in similar increases in the magnitude of downstream responses (Law et al., 1994; Prather et al., 1994). Third, the relative differences in efficacy among agonists also depended on receptor density. Similar results have been observed in other receptor systems at the level of both the effector (Boddeke et al., 1992; MacEwan et al., 1995) and G-protein (Newman-Tancredi et al., 1997). Fourth, the unique finding of the present study was that the combination of the apparent K_D and B_max of net agonist-stimulated [³⁵S]GTPS binding correlated with intrinsic agonist efficacy for G-protein activation. All these results support hypotheses proposed in our previous studies of mu opioid agonist efficacy (Selley et al., 1997). These studies showed that opioid mu agonists may be divided into three classes of efficacy based on apparent K_D and B_max values from saturation analysis of agonist-stimulated [³⁵S]GTPS binding. By this classification, full agonists produce both the lowest K_D values and the highest B_max values, whereas typical partial agonists produce both higher K_D values and lower B_max values, and "mixed full/partial" or "high efficacy partial" agonists produce the lowest K_D values (like full agonists), but lower B_max values (like partial agonists). This last class of agonists may appear to be full or partial depending on the tissue, as observed with morphine, for example, in mMOR-CHO cells versus rat thalamus.

These results raise an important question: What factors determine whether differences in intrinsic efficacy for G-protein activation are expressed as differences in the maximal stimulation (%_Max) or in the K_i/EC₅₀ ratio (receptor reserve)? Clearly, the lower apparent affinity of net-stimulated [³⁵S]GTPS binding produced by classical partial agonists (such as buprenorphine) resulted in lower %_Max values in all systems examined so far. Similarly, the lowest affinities of net-stimulated [³⁵S]GTPS binding in mMOR-CHO membranes were observed with the lowest efficacy partial agonists (such as nalbuphine and levallorphan), which all tended to act as full (neutral) antagonists in rat thalamic membranes under the conditions tested. In contrast, those drugs (such as morphine) which were full agonists in mMOR-CHO cells, but partial agonists in rat thalamus, produced K_D values for [³⁵S]GTPS binding similar to those produced by DAMGO in each system. However, they generally produced B_max values that differed from DAMGO only in thalamic membranes. Thus, one possible explanation for the differences in maximal stimulation observed in thalamic membranes is that there were not enough receptors present in this system to fully activate the available G-protein pool. This is supported by the lack of receptor reserve for G-protein activation in this system, as indicated by K_i/EC₅₀ values that were not greater than one. In mMOR-CHO cell membranes, however, the number of receptors may have been sufficient to fully activate the pool of G-proteins that were available for coupling to mu opioid receptors, resulting in receptor reserve for G-protein activation by most of the full agonists in this system. Complete activation of the available G-protein pool would explain the "ceiling effect," whereby agonists that produced different levels of stimulation in thalamic membranes (such as DAMGO and morphine) produced the same maximal stimulation in mMOR-CHO membranes. This possibility is supported by the reported density of inhibitory G-protein  subunits in CHO cells: ~5.5 pmol/mg (Gettys et al., 1994b), which is very close to the 3.5 to 4 pmol/mg of net-stimulated [³⁵S]GTPS binding obtained in the presence of maximal stimulatory concentrations of full mu agonists in mMOR-CHO membranes.

The hypothesis that differences in receptor density can account for the differences in relative agonist efficacy is supported by previous studies in 5-HT_1A receptor-transfected cells, where differences in relative agonist efficacies were observed at the effector level between cells expressing 0.5 versus 3 pmol receptor/mg (Boddeke et al., 1992), but not between those expressing 0.05 versus 0.5 pmol receptor/mg (Varrault et al., 1992). Similarly, an increase in the relative efficacy of a partial agonist for stimulation of [³⁵S]GTPS binding has been observed in CHO cells expressing 5-HT_1A receptors at a density of 4.2 pmol/mg versus 1.6 pmol/mg (Newman-Tancredi et al., 1997). These studies suggest that there may be a minimal receptor expression level (~1 pmol/mg) above which differences in relative efficacy are minimized. This is supported by our studies in the mu opioid system, where we previously have observed differences in relative agonist efficacies for G-protein activation between rat thalamus and mMOR-CHO cells (0.75 versus 6.5 pmol receptor/mg, respectively), but not between SK-N-SH cells and rat thalamus (0.15 versus 0.75 pmol receptor/mg, respectively) (Selley et al., 1997). Although SK-N-SH cells contained a 5-fold lower density of mu receptors than rat thalamus, saturation analysis of agonist-stimulated [³⁵S]GTPS binding revealed that the B_max of activated G-proteins was also approximately 5-fold lower in these cells than thalamus. This finding suggests that it may actually be the ratio of receptors to activated G-proteins that determines relative differences in maximal stimulation. This point is illustrated by examining the amplification factor, or number of G-proteins activated per receptor, among the three systems. As shown in table 3 (with use of the full agonist DAMGO), the amplification factors in thalamic and SK-N-SH membranes are identical with each other, but are approximately 20-fold higher than in mMOR-CHO membranes. These results suggest that the receptor/transducer ratio and the resulting amplification factor may be important in determining how differences in agonist intrinsic efficacy are expressed as functional responses. A similar conclusion was reached in the 5-HT_1A system, where increasing the receptor density did not result in an increased number of activated G-proteins, but did increase the relative efficacy of a partial agonist (Newman-Tancredi et al., 1997). Thus, the receptor/G-protein ratio may determine the critical range of receptor B_max values within which efficacy differences are maximized.

View this table: [in this window] [in a new window]   TABLE 3 Amplification factors for mu opioid receptor-stimulated G-protein activation in cultured cell lines and rat thalamus Receptor and [35S]GTPS binding assays for saturation analysis were conducted under identical conditions, as described under "Materials and Methods." The mu receptor Bmax was determined by [3H]naloxone binding, and the G-protein Bmax was determined by net DAMGO-stimulated [35S]GTPS binding. Amplification factor defined by DAMGO-activated G-protein Bmax/mu receptor Bmax.

Another important factor is the level of the signal transduction pathway at which efficacy determinations are made. Clearly, high-efficacy agonists such as DAMGO, methadone, morphine, fentanyl and sufentanil, which showed no receptor reserve for G-protein activation in rat thalamic membranes, display receptor reserve for the production of biological responses in whole animals (Adams et al., 1990; Mjanger and Yaksh, 1991; Zernig et al., 1994, 1995) and isolated organ preparations (Chavkin and Goldstein, 1984; Ivarsson and Neil, 1989). It is interesting that in assays of mu receptor-mediated inhibition of adenylyl cyclase activity, high-efficacy partial agonists, such as morphine, tend to show 90% of the maximal response produced by the full agonist DAMGO. Similarly, low-efficacy partial agonists, such as nalorphine, nalbuphine and levallorphan, also showed greater maximal inhibition of adenylyl cyclase (35-40% of DAMGO) than was observed for G-protein activation in the present study (Yu and Sadee, 1988; Carter and Medzihradsky, 1993). One explanation for these differences is that the adenylyl cyclase assay conditions favor G-protein activation: relatively high (µM) GTP concentrations are used with no added GDP. Such conditions should favor the relative efficacy of partial agonists because high GDP concentrations tend to amplify and low GDP concentrations tend to minimize efficacy differences among agonists (Lorenzen et al., 1996; Selley et al., 1997). Another factor which may influence measurements of relative efficacy at different levels of the signal transduction pathway is that the effector may be the limiting step: nonadditive modulation of effectors has been observed between receptors that produce additive stimulation of G-protein activity (Andrade et al., 1986; Pacheco et al., 1993; Okuhara and Beck, 1994; Odagaki and Fuxe, 1995). Thus, the number of G-proteins available for receptor activation generally may exceed the effector systems to which they couple. This is supported by the observation that there is delta opioid receptor reserve for adenylyl cyclase inhibition in the absence of receptor reserve for low K_m GTPase stimulation in NG108-15 cells (Costa et al., 1988). Additionally, there may be different levels of receptor reserve for different G-protein-coupled effector responses (Boddeke et al., 1992). Thus, measurements of agonist intrinsic efficacy at the level of receptor-G-protein activation are likely to be the most accurate indicator of this parameter, as suggested previously (Keen 1991).

Another issue not directly addressed in detail in the present study is the relationship between high- and low-affinity conformations of the receptor binding site and the production of functional responses. Binding K_i values in the present study were approximately 100-times higher than the high-affinity K_i values reported in the literature for most agonists (Corbett et al., 1993; Emmerson et al., 1994). Although these K_i values were consistent with agonist binding primarily to a low-affinity conformation of the receptors, many agonists displayed Hill coefficients less than one for receptor binding in both mMOR-CHO cell and rat thalamic membranes. In general, higher efficacy agonists tended to show lower Hill slopes, although the correlation between intrinsic efficacy and Hill slope was not significant. Moreover, not only was there no detectable receptor reserve for G-protein activation in thalamus, but the K_i/EC₅₀ ratios tended to be less than one. Although this trend was below the level of significance for all but one agonist (methadone), a K_i/EC₅₀ ratio of less than one would suggest that an agonist would have to occupy more receptors than are present to produce the corresponding functional response (a theoretical impossibility). One explanation is that the EC₅₀ values corresponded to the binding K_i values for the low-affinity form of the receptors. This possibility was tested in detailed binding displacement curves with methadone in rat thalamus (data not shown), where the K_i value for the low-affinity site was approximately 4-fold higher than the EC₅₀ for stimulation of [³⁵S]GTPS binding, and the high-affinity K_i was more than 25-fold lower than the EC₅₀. Thus, it is possible that there is a small receptor reserve for this full agonist at its low-affinity binding site in thalamic membranes. Alternately, both the high- and low-affinity binding sites could contribute unequally, with the agonist-induced response primarily being caused by its action at low-affinity "uncoupled" receptors, which are then "driven" to couple with G-proteins by agonist occupation. High-affinity sites may represent precoupled or spontaneously active forms of the receptor (Costa et al., 1990; Tian et al., 1994), which may be recognized by some agonists with high affinity. These high-affinity receptors may activate G-proteins in the absence of agonist and contribute to basal rather than agonist-stimulated [³⁵S]GTPS binding. A detailed examination of this relationship is beyond the scope of the present study and will be addressed in future investigations. It is clear, however, from the present study that a simple K_i/EC₅₀ ratio may somewhat underestimate the true receptor reserve when the Hill coefficient of the receptor binding curve is less than one. Nevertheless, the consistency of this error among most full agonists indicates that this measurement is at least proportional to the true receptor reserve.

Another important finding is that most, but not all, agonists showed relative intrinsic efficacy values that corresponded to previous reports of opioid agonist efficacy in other systems. Two other groups (Traynor and Nahorski, 1995; Emmerson et al., 1996) who reported the use of agonist-stimulated [³⁵S]GTPS binding to measure mu agonist efficacy have found qualitatively similar results. However, both of these previous studies reported a higher maximal stimulation by fentanyl and/or sufentanil relative to morphine. This agrees with many studies which show a higher intrinsic efficacy of fentanyl (Adams et al., 1990; Zernig et al., 1995), sufentanil (Mjanger and Yaksh, 1991) and alfentanil (Zernig et al., 1994) relative to morphine in antinociception paradigms. We can not explain these apparent discrepancies regarding the efficacy of the fentanyl-derived compounds relative to morphine. However, not all animal models of antinociception show a significant efficacy difference between fentanyl and morphine (T.J. Martin, Wake Forest Univ. Sch. Med., 8/15/97, personal communication). Moreover, when the K_i/EC₅₀ ratio was taken into account in the report by Emmerson et al. (1996), DAMGO was found to be much more efficacious than either morphine or sufentanil, and morphine was found to be somewhat more efficacious than sufentanil, which agrees more with the results of the present study. Furthermore, higher intrinsic efficacy measurements of DAMGO and methadone relative to morphine have been reported in assays of antinociception in rodent models (Adams et al., 1990; Mjanger and Yaksh, 1991; Zernig et al., 1995) and in bioassays in the guinea pig ileum (Ivarsson and Neil, 1989), in agreement with the present study. Moreover, this study and a previous report from our laboratory (Selley et al., 1997) are the only studies that have examined the relative efficacy of these agonists for G-protein activation in rat brain, as well as in mMOR-CHO and SK-N-SH cells. All three systems have shown no significant efficacy difference between morphine and the fentanyl-related compounds.

Further studies will be required to elucidate all the relevant factors that influence measurements of relative agonist efficacy. For example, the relative levels of the various G-protein  subunit subtypes to which the receptor couples also may influence relative efficacy, because receptors may differentially activate different G-proteins depending on the specific agonist used (Kenakin and Morgan, 1989; Gettys et al., 1994a; Kenakin, 1996). For mu opioid receptors, coupling to multiple G-protein subtypes has been demonstrated in various cell types (Laugwitz et al., 1993; Chakrabarti et al., 1995). Cell-specific factors in addition to the overall receptor/G-protein ratio, including both quantitative and qualitative parameters of receptor-G-protein interaction, may influence the relative intrinsic efficacy of agonists.

In conclusion, the present study has shown that the intrinsic efficacy of agonists acting at mu opioid receptors correlates with both the maximal number of G-proteins activated by the agonist-occupied receptor and with the apparent affinity with which these activated G-proteins bind the GTP analog. Whether differences in agonist intrinsic efficacy are expressed as differences in receptor reserve or in maximal response depended on the number of receptors relative to the number of available G-proteins; where R > G, receptor reserve for G-protein activation was observed. These results predict that relative differences in the maximal responses produced by agonists will depend on the intrinsic efficacy of the agonist and the receptor/G-protein ratio, in addition to the level of the signal transduction pathway at which the response is measured and the relative number of the different types of G-proteins (and the respective effectors) to which the receptor can couple.