Abstract
The ability of endogenous opioids to activate G proteins was measured in membranes from C6 rat glioma cells stably expressing a cloned rat mu receptor. Peptides representing each of the three known families of endogenous opioids (enkephalins, endorphins and dynorphins) were studied, as well as two recently discovered endogenous opioids, endomorphin-1 and -2, which are thought to represent a fourth family of endogenous opioid peptides. Stimulation of guanosine-5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding to membranes was used as a measure of G protein activation. It was possible to differentiate high efficacy compounds such as Tyr-d-Ala-Gly-(Me)Phe-Gly-ol from lower-efficacy agonists such as morphine or meperidine. Met- and leu-enkephalin, beta endorphin and dynorphin A were all found to have high efficacy at the mu receptor, as were the peptide fragments beta endorphin-(1–27) and dynorphin A-(1–13). Endomorphin-1 and -2 were found to be partial agonists, capable of both stimulating [35S]GTPγS binding and antagonizing the stimulation produced by the higher-efficacy agonist Tyr-d-Ala-Gly-(Me)Phe-Gly-ol. Binding affinities for the opioid agonists at the cloned mu receptor were measured by the displacement of radiolabeled antagonist. It was found that theKi values closely matched the EC50 values for [35S]GTPγS binding stimulation, indicating that a large receptor reserve does not exist for the complete activation of G proteins in this system.
Endogenous opioid peptides regulate many physiological functions. Best known for their ability to modulate the sensation of pain (Akil et al., 1984), endogenous opioids also regulate gastrointestinal motility (Kromer, 1988), the production and release of neuroendocrine hormones and immune system function (e.g.,Herz, 1993). Endogenous opioids are also believed to play a role in mediating reinforcement, and may be involved in modulating behaviors such as eating and drinking (e.g.,Holtzman, 1975), some aspects of sexual behavior (e.g.,Pfaus and Gorzalka, 1987; Gessa et al., 1979) and the abuse of alcohol (e.g.,Gianoulakis and Waele, 1994).
Many endogenous opioid peptides have been identified, and they have been divided into three major families based on the large precursor molecules from which they are enzymatically cleaved (Akil et al., 1988). The endorphins, enkephalins and dynorphins are derived from proopiomelanocortin, proenkephalin and prodynorphin, respectively. Additionally, two recently described endogenous opioid peptides, endomorphin-1 (Tyr-Pro-Phe-Phe-NH2) and endomorphin-2 (Tyr-Pro-Trp-Phe-NH2), have been discovered in bovine brain (Zadina et al., 1997). These peptides are interesting because it can be inferred from their amino acid sequence that they are not derived from any previously known precursor molecules, and therefore may represent a “new” family of endogenous opioids. The endomorphins are reported to have very high affinity and selectivity for the mu receptor and have been postulated to be “the” endogenous mu selective ligands. It is therefore of interest to characterize the activity of these peptides at the mu receptor in vitro. Our study is designed to determine the extent to which the endomorphins and representative peptides from each of the other three families of endogenous opioids are able to activate the mu opioid receptor.
Information about the activity of some endogenous opioids at themu receptor has been previously described. For example,Kapas et al. (1995) found both met- and leu-enkephalin to be agonists at the mu receptor in primary cultures of rat zona glomerulosa cells. Chavkin et al. (1985), using selective opioid antagonists in rat hippocampal slices, concluded that dynorphin A and B may be agonists at the mu receptor. The truncated peptide, dynorphin A-(1–13), however, has been shown to antagonizemu receptor-mediated analgesia in rhesus monkeys (Butelman,et al., 1995). Similarly, although full lengthbeta endorphin [beta endorphin-(1–31)] is generally accepted as a high efficacy mu agonist, the naturally occurring cleavage fragment beta endorphin-(1–27) is reported to be a partial agonist capable of antagonizingbeta endorphin- and etorphine-induced analgesia in the mouse tail-flick assay (Hammonds et al., 1984; Nicolas and Li, 1985). However, the efficacies of beta endorphin-(1–27) and full length beta endorphin have not been previously comparedin vitro.
Although the experiments described above provide information about which endogenous peptides are likely to have some activity at themu opioid receptor, no attempt has been made to quantitatively compare the relative efficacies of these peptides to each other or to the exogenous drugs that act through the same receptor system. Drug efficacy is defined by Stephenson (1956) as a property of the agonist at a given receptor that remains constant independent of tissue or preparation. Therefore an appropriate determination of the relative efficacies of the endogenous opioids at the mureceptor should provide information that can be generalized to predict how they will act at the mu receptor in other situations. The current study is designed to provide this information by measuring the ability of several endogenous opioids to activate G proteins through the mu receptor. To do this we have assessed the binding of the GTP analog [35S]GTPγS after agonist occupation of the receptor.
Opioid receptors belong to the seven transmembrane superfamily of heterotrimeric guanine nucleotide-binding protein- (G protein) coupled receptors, and are linked to the adenylyl cyclase-inhibitory G proteins Gi and Go (Carter and Medzihradsky, 1993). In the resting state, the α subunit of the G protein is bound to GDP. Activation of the receptor by an agonist leads to the dissociation of GDP from the G protein, allowing GTP to bind (Higashijima et al., 1987; Gilman, 1987). This leads to the dissociation of the α and βγ subunits of the G protein, which are then able to interact with effector systems. The binding of the nonhydrolyzable GTP analog [35S]GTPγS has been previously used to provide a measure of G protein activation by agonists (e.g.,Lorenzen et al., 1993; Tianet al., 1994). Because G protein activation is the first biochemical step after opioid receptor activation and is not limited by downstream effector systems, this assay provides a very direct measurement of efficacy. Traynor and Nahorski (1995) and Emmersonet al. (1996) have demonstrated the usefulness of this assay for determining the relative efficacies of mu opioid agonists in vitro, using exogenous drugs that have been well characterized. The correlation between the intrinsic activity of a drug in this assay and its efficacy in vivo makes it an appropriate system for measuring the relative efficacies of the endogenous opioid peptides, which are not well characterized. The current study uses the [35S]GTPγS binding assay to assess the ability of endogenous opioids to activate G proteins in a rat glioma cell line (C6) expressing a cloned rat mu receptor (Thompson et al., 1993). Because the C6 cell line does not express any opioid receptors other than the transfectedmu receptor, this system is free from the potential contribution of delta or kappa opioid receptor activation.
Methods
Materials.
DAMGO, leu-enkephalin, met-enkephalin, PMSF, EDTA, iodoacetamide, bestatin, thiorphan, captopril and l-leucyl-leucine were obtained from Sigma Chemical Co. (St. Louis, MO). Naloxone was provided by Du Pont (Wilmington, DE). Morphine was purchased from Mallinckrodt (St. Louis, MO). Meperidine was provided by the Sterling-Winthrop Research Institute (Rensselaer, NY). Sufentanil was a gift from Janssen (New Brunswick, NJ). Betaendorphin-(1–27) and beta endorphin-(1–31) were purchased from Peninsula Laboratories (Belmont, CA). Dynorphin A-(1–17), dynorphin A-(1–13) and dynorphin A-(2–13) were provided by Chiron Multiple Peptide Systems (San Diego, CA). U–50488H was provided by Upjohn (Kalamazoo, MI). Endomorphin-1 and -2 were purchased from American Peptide Company (Sunnyvale, CA). Aprotinin, leupeptin and pepstatin were from Boehringer Mannheim (Indianapolis, IN). [35S]GTPγS (specific activity 1250 Ci/mmol) and [3H]naloxone (specific activity 58.5 Ci/mmol) were purchased from Du Pont NEN (Boston, MA). Dulbecco’s medium and other biochemicals were from Sigma. Fetal bovine serum and Geneticin were purchased from Gibco Life Sciences (Gaithersberg, MD).
Cell culture.
C6(μ) rat glioma cells stably transfected with a rat mu opioid receptor (Emmersonet al., 1996) were grown under 5% CO2in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Stock flasks were maintained in the presence of 1 mg/ml Geneticin to select for the presence of the transfected plasmid, which codes for both the mu receptor and antibiotic resistance. Cells used for experiments were split from the stock flasks and grown to confluence in the absence of Geneticin without significant loss in receptor density.
Membrane preparation.
Cells were rinsed twice with ice-cold phosphate-buffered saline (0.9% NaCl, 0.61 mM Na2HPO4, 0.38 mM KH2PO4, pH 7.4) and detached from dishes by incubation with lifting buffer (5.6 mM glucose, 5 mM KCl, 5 mM HEPES, 137 mM NaCl, 1 mM EGTA, pH 7.4). The cells were then pelleted and resuspended in ice-cold lysis buffer (0.2 mM MgSO4, 0.38 mM KH2PO4, 0.61 mM Na2HPO4, pH 7.4) and homogenized using a glass-glass Dounce homogenizer. Membranes were isolated by centrifugation for 20 min at 20,000 × g at 4°C. The resulting membrane pellets were re-suspended in 50 mM Tris buffer (pH 7.4) and stored at -80°C in 1-ml aliquots (approximately 1 mg protein/ml).
Protein determination.
Protein concentration in membrane samples was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard. Samples were solublized by incubation at room temperature in 0.5 N NaOH for 30 min before protein determination.
[35S]GTPγS binding assay.
Varying concentrations of ligand were preincubated with membranes (15 μg membrane protein/tube) for 2 hr at 25°C in binding cocktail [30 μM GDP, 1 mM dithiothreitol, I mM EDTA, 5 mM MgCl2, 100 mM NaCl and 47 mM Tris (pH 7.4)] in a 200 μl final assay volume. Experiments were initiated by the addition of [35S]GTPγS (final concentration 40 pM), which was added in a volume of 10 μl H2O, so as to cause only a small (<5%) change in the concentration of ligand and other reagents. After 1 min the reaction was terminated by the addition of 2 ml ice-cold washing buffer (50 mM Tris, 5 mM MgCl2, 100 mM NaCl) and the contents of the tubes were rapidly filtered through glass fiber filters (Schleicher & Schuell no. 32, Keene, NH). The tubes and filters were then rinsed with 2 ml washing buffer an additional three times. Filters were placed in scintillation vials containing 400 μl ethanol and 4-ml scintillation cocktail for liquid scintillation counting. Nonspecific counts were determined from tubes which contained 100 nM unlabeled GTPγS.
Receptor binding assay.
Saturation binding experiments were performed using [3H]naloxone. For these experiments, the radioligand was added to membranes (10 μg protein/tube) in binding cocktail (see [35S]GTPγS binding assay) which contained 40 pM unlabeled GTPγS in order to control for any effect which the nucleotide may have on ligand affinity. Assay volume was 2 ml. Samples were incubated for 2 hr at 25°C, quickly filtered through glass fiber filters (Schleicher & Schuell no. 32) mounted on a Brandell cell harvester, washed three times with ice-cold 50 mM Tris buffer (pH 7.4), and subjected to liquid scintillation counting. Nonspecific counts were determined from samples that contained 10 μM unlabeled naloxone. Displacement of radiolabeled [3H]naloxone (3 nM) by varying concentration of unlabeled drug used the same procedure.
Data analysis.
The Graph Pad Prism computer program (San Diego, CA) was used to perform linear and nonlinear regression analysis of the data. [35S]GTPγS binding data was fitted to a sigmoidal curve with variable slope, with the baseline value fixed at 0% stimulation. Displacement curves were fitted to a one-site competition curve with top and bottom fixed at 100% and 0% of control radioligand binding, respectively. Dissociation constants (Ki
) were calculated according to the Cheng-Prusoff equation:
The Kd value for naloxone, as determined from rightward shifts in agonist concentration-response curves, was calculated according to the Schild formula using a single concentration of competitive antagonist (Kosterlitz and Watt, 1968):
Statistics.
Differences between the intrinsic activity of different agonists were analyzed for significance using Tukey’s honest significant difference test for unequal n, with the aid of the STATISTICA for Windows computer program from StatSoft, Inc. (Tulsa, OK).
Results
It has been previously demonstrated that agonist stimulation of [35S]GTPγS binding in C6(μ) membranes requires the presence of both Na+ and GDP, is stereoselective, and is blocked by the addition of pertussis-toxin (Emmerson et al., 1996). It was determined from pilot experiments that partial agonists could be more easily differentiated from full agonists at shorter [35S]GTPγS incubation periods; therefore our study used a 1-min incubation with [35S]GTPγS, after a 2-hr preincubation of membranes with agonist to allow agonist binding to reach equilibrium. Under these conditions the mu agonist DAMGO concentration-dependently increased the binding of [35S]GTPγS by 14.4 ± 0.8 fmol/mg of membrane protein from a basal level of 0.4 ± 0.3 fmol/mg protein (fig. 1). Addition of the competitive antagonist naloxone produced a rightward shift in the DAMGO concentration-response curve, yielding aKd value for naloxone of 1.56 nM according to the Schild formula (see “Methods”). TheKd of [3H]naloxone was also determined in saturation binding assays. [3H]naloxone bound to C6(μ) membranes withKd = 1.29 ± 0.11 nM and Bmax = 2.77 ± 0.24 pmol/mg protein (fig.2).
The ability of endogenous opioids to stimulate [35S]GTPγS binding was measured and compared to the stimulation produced by DAMGO and the nonpeptide agonists sufentanil, morphine and meperidine. The “intrinsic activity” of opioid agonists at the mu receptor is indicated by the maximum stimulation of [35S]GTPγS binding attainable. This value is given relative to the maximum effect produced by DAMGO (fig. 3; table1). Met-enkephalin, leu-enkephalin,beta endorphin-(1–31) and beta endorphin-(1–27) were full agonists, producing 98 to 117% of the stimulation of [35S]GTPγS binding produced by DAMGO, with potencies: DAMGO (EC50 = 145 nM) > met-enkephalin > beta endorphin-(1–31) >beta endorphin-(1–27) > leu-enkephalin (EC50 = 956 nM). Sufentanil was an intermediate efficacy agonist, producing 66% of DAMGO’s maximum effect, with high potency (EC50 = 3 nM). In this assay morphine and meperidine produced about the same maximum effect, 41 and 45% of DAMGO stimulation, respectively. However, although morphine had relatively high potency (EC50 = 118 nM), meperidine had extremely low potency (EC50 = 22 μM). Concentration-response curves for dynorphin A-(1–17) and dynorphin A-(1–13) were limited by nonspecific effects on membrane viscosity which were seen at concentrations higher than 1 μM. However, enough of the concentration-effect curves for these peptides could be determined to show that both have relatively high efficacy at themu receptor, having intrinsic activities higher than those of meperidine or morphine. Dynorphin A-(2–13), which is missing the N-terminal Tyr residue believed to be critical for opioid receptor binding, was completely ineffective as an agonist in this assay, as was the kappa selective agonist U–50488H. Endomorphin-1 and -2 were both partial agonists under the conditions used, producing 65 and 63% of maximum DAMGO stimulation, respectively, with potencies similar to the other endogenous ligands (EC50 = 293 nM for endomorphin-1, EC50 = 521 nM for endomorphin-2).
Concentration-effect curves for peptides were also examined in the presence of two different “cocktails” of enzyme inhibitors (0.1 mM PMSF, 1 μg/ml aprotinin, 1 mM EDTA, 1 μg/ml leupeptin, 1 μg/ml pepstatin and 1 mM iodoacetamide; or 10 μM bestatin, 0.3 μM thiorphan, 10 μM captopril and 2 mM l-leucyl-leucine). Neither of these cocktails had any effect on the concentration-response curves for the peptides (data not shown), implying that enzymatic peptide cleavage does not play a significant role under the conditions of this assay.
Differences in the intrinsic activity measured for each drug were analyzed for significance according to Tukey’s honest significant difference test for unequal n (table 2). No significant differences were seen among met-enkephalin, leu-enkephalin, beta endorphin-(1–31) and betaendorphin-(1–27), although all had intrinsic activity significantly higher than morphine and meperidine. Endomorphin-1 and endomorphin-2 both had intrinsic activity significantly lower than leu-enkephalin andbeta endorphin-(1–31). Sufentanil’s maximum effect was significantly different from only leu-enkephalin. In general, the pattern of significance is consistent with the interpretation that met-enkephalin, leu-enkephalin, beta endorphin-(1–31) andbeta endorphin-(1–27) are all full agonists, and sufentanil, morphine, meperidine and endomorphin-1 and -2 have lower efficacy at this receptor.
To test the assumption that beta endorphin-(1–31) andbeta endorphin-(1–27) produced their effects through the same receptor population as DAMGO, concentration-response curves were also determined for these peptides in the presence of the competitive antagonist naloxone. Naloxone produced surmountable rightward shifts in the concentration-response curves for both betaendorphin-(1–31) and beta endorphin-(1–27) (fig.4), which were similar to the effect of naloxone on the DAMGO concentration-response curve (fig.1). Calculation of naloxone’s affinity based on the rightward shifts produced for beta endorphin-(1–31) and betaendorphin-(1–27) yielded Kd values of 2.22 nM and 1.23 nM, respectively, values consistent with equivalent receptor involvement.
In contrast to the other endogenous opioid peptides studied, endomorphin-1 and -2 were weak agonists in this system, producing 63 to 65% of maximum DAMGO stimulation. This is hypothesized to reflect that the endomorphins have less efficacy at the mu receptor than DAMGO and the other endogenous ligands (table 2). Therefore the ability of endomorphin-2 to antagonize a full agonist, DAMGO, was tested (fig.5). Endomorphin-2 reduced the stimulation produced by 10 μM DAMGO in a concentration-dependent manner to 61% of the maximum effect produced by DAMGO alone.
The affinity of the ligands for the mu receptor was also determined by displacement of [3H]naloxone under conditions identical to those used for the [35S]GTPγS binding assay (table 1). All of the endogenous ligands tested showed similar affinity for this receptor in the order: Met-enkephalin (Ki = 179 nM) > β-endorphin > endomorphin-2 > endomorphin-1 = dynorphin A-(1–13) > dynorphin A-(1–17) = leu-enkephalin (Ki = 779 nM). The exogenous drugs tested exhibited a much greater range of affinities: sufentanil showed extremely high affinity (Ki = 4 nM); meperidine had very low affinity (Ki = 35 μM); DAMGO and morphine both had affinities comparable to the endogenous ligands (Ki = 185 nM and 195 nM, respectively). In all cases the affinity (Ki value) measured in the displacement assay closely matched the potency (EC50 value) determined in the [35S]GTPγS binding assay. The correlation of pEC50 with pKi for the agonists tested was r2 = 0.972 with slope of 0.988. This indicates that no significant receptor reserve exists for the activation of G proteins in this system.
Discussion
Opioid agonists increased the binding of [35S]GTPγS in membranes from C6 rat glioma cells stably expressing the ratmu opioid receptor. Agonist stimulation of [35S]GTPγS binding is concentration-dependent, stereoselective, pertussis-toxin sensitive, and surmountably antagonized by the competitive opioid antagonists naloxone and naltrexone (present study and Emmerson et al., 1996). The intrinsic activity of agonists in this assay has been shown to correlate with the efficacy of these drugs observed in vivo (Emmerson et al., 1996). This was also observed in our study, in which high-efficacy agonists, such as DAMGO, could be distinguished from lower-efficacy agonists such as morphine (Adamset al., 1990; Comer et al., 1992). In fact it was possible in this assay to differentiate sufentanil from higher-efficacy agonists. Although sufentanil is a full agonist in vivo, it has been previously reported to be a lower-efficacy agonist in vitro (Emmerson et al., 1996). Because in our study all ligands were preincubated with membranes to allow receptor binding to reach equilibrium, the lower efficacy measured for sufentanil is unlikely to be an artifact produced by slow binding kinetics. This interesting discrepancy needs further study.
All endogenous opioids tested were agonists at the rat mureceptor. Most were full agonists. This was most surprising in the case of beta endorphin-(1–27), which had been predicted to be a partial agonist based on in vivo data (see below). Also surprising was the finding that endomorphin-1 and endomorphin-2, the peptides that have been postulated to represent the primary endogenous ligands for the mu receptor, actually had the lowest efficacy of all endogenous ligands tested at the mureceptor.
Met- and leu-enkephalin were both full agonists, producing 97 and 117% of DAMGO’s maximum effect, respectively. This is consistent with earlier findings by Kapas et al. (1995) that both met- and leu-enkephalin produce mu receptor activation in primary cultures of rat zona glomerulosa cells.
Dynorphin A-(1–17) and dynorphin A-(1–13) also appear to be relatively high-efficacy agonists at the mu receptor in this assay. This interpretation of the results is strengthened by the fact that neither dynorphin A-(2–13) nor the kappa selective agonist U–50488H had any agonist effects in this assay. The lack of stimulation of [35S]GTPγS binding by U–50488H confirms that the effects of the dynorphins are not mediated through kappa receptors. The lack of an agonist effect of dynorphin A-(2–13), which is missing the N-terminal Tyr critical for opioid receptor binding, supports the idea that the dynorphins are acting through the mu receptor rather than through some nonreceptor-related mechanism. The finding that dynorphin A-(1–17) is a mu agonist is supported by an earlier study by Chavkinet al. (1985), in which dynorphin A-(1–17) was found to be an agonist at the mu receptor in rat hippocampal slices.
The efficacy of dynorphin A-(1–13) in the present assay, however, is in apparent disagreement with a report by Butelman et al. (1995) that dynorphin A-(1–13) antagonizes mureceptor-mediated analgesia in rhesus monkeys, implying that dynorphin A-(1–13) has little or no efficacy at the mu receptor. It is possible that this difference may be explained by differences in the structure of rat and monkey mu receptors. It may also be likely that the dynorphin A-(1–13) antagonism of in vivo mu effects represents physiological antagonism rather than competitive antagonism at the mu receptor. Physiological antagonism of mu effects by kappa agonists has been reported previously; Itoh et al. (1994) showed that dynorphin A-(1–13) could antagonize effects of mu receptor agonists in mice, but this antagonism was blocked by thekappa selective antagonist nor-binaltorphamine.
Human beta endorphin-(1–31) and betaendorphin-(1–27) were also full agonists in stimulating [35S]GTPγS binding, producing 110 and 98%, respectively, of DAMGO’s maximum effect. The competitive antagonist naloxone produced rightward shifts in the concentration-effect curves for these peptides which were of approximately the same magnitude as the shift produced for DAMGO, indicating that the [35S]GTPγS binding stimulation produced by the endorphins is mediated through the mu receptor. Although it was not surprising that beta endorphin-(1–31) was a full agonist at the mu receptor, Li and colleagues (Hammondset al., 1984; Nicolas and Li, 1985) have found that humanbeta endorphin-(1–27) was capable of antagonizing etorphine-induced analgesia in mice. The antagonism produced bybeta endorphin-(1–27) in these in vivoexperiments was dose-dependent, and appeared, based on pA2 analysis, to represent competitive antagonism. Therefore the possibility of physiological antagonism mediated through another receptor type, such as seen with dynorphin A-(1–13), seems less likely to explain the discrepancy between these reports and the current findings. The possibility that betaendorphin-(1–27) could be efficacious in the present experiment because it is being cleaved to a more active peptide, such as met-enkephalin, also seems unlikely since the concentration-effect curve for beta endorphin-(1–27) was not affected by the presence of enzyme inhibitors. Conversely, it may be possible thatbeta endorphin-(1–27) is more susceptible than full-lengthbeta endorphin to cleavage in vivo. Such cleavage may be to a form that retains affinity, but not activity, at themu receptor. Also, there is again always the possibility that these conflicting results may be explained by species differences or other differences between the cloned rat mu receptor and the receptor population that mediates the effects of betaendorphin and etorphine in mouse brain.
Interestingly, the endomorphins, which are reported to exhibit greater selectivity for the mu receptor than the other endogenous opioids (Zadina et al., 1997), were less efficacious than the other endogenous agonists under the current conditions, producing about 63 to 65% of the maximum response produced by DAMGO. The notion that these peptides are of lower efficacy at the mu receptor is supported by the ability of endomorphin-2 to antagonize the [35S]GTPγS binding stimulation produced by DAMGO. Endomorphin-1 has been shown to be a mu agonist in vivo in mice, capable of producing a maximal effect in the focused light tail-flick assay (Zadina et al., 1997). It should be noted however that morphine is also able to produce a maximal response in that assay.
For all of the drugs tested, the EC50 values for [35S]GTPγS binding stimulation closely matched the Ki values determined under identical conditions. In other words, roughly the same concentration of drug that occupies half of the receptors is needed to produce a half-maximal response. This shows that a large spare receptor population does not exist for the activation of G proteins in this assay. This may reflect an inability of individual G proteins to be activated by multiple mu receptors. It should be noted that this does not imply that individual receptors cannot activate multiple G proteins, allowing for “amplification” of the signal at this level. Spare receptors are likely to exist for downstream effector systems, such as adenylyl cyclase (Fantozzi et al., 1981;Law et al., 1982). The lack of a receptor reserve in this assay means that the intrinsic activity, or maximum effect producible by each of the drugs, can be interpreted as a direct measurement of efficacy, making this system especially suitable for measuring agonist efficacy. More information is needed to determine whether the apparent lack of spare receptors seen in this study reflects normal cellular functioning in vivo or if the appearance of spare receptors is somehow limited by the artificial nature of the system used. For instance, there may be differences between transfected and endogenously expressed opioid receptors which are not yet appreciated. Future experiments should be designed to answer these questions and further elucidate how the results of the current study pertain to the actions of the endogenous opioids in vivo.
Acknowledgments
The authors thank Dr. Richard Neubig for advice and comments and Brandy Johnson, Eunice Hong and Young Mee Rhee for technical assistance.
Footnotes
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Send reprint requests to: Andrew Alt, 1301 Medical Science Research Building III, The University of Michigan Medical School, Department of Pharmacology, Ann Arbor, MI 48109-0632.
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↵1 This work was supported by National Institutes of Health Grants R01 DA04087, R01 DA02265, DA00254, T32-DA07281 and T32-GM07767
- Abbreviations:
- G protein
- GTP-binding protein
- GTP
- guanosine triphosphate
- GDP
- guanosine diphosphate
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- cAMP
- adenosine 3′,5′-cyclic monophosphate
- DAMGO
- Tyr-d-Ala-Gly-(Me)Phe-Gly-ol
- Tris
- tris(hydroxymethyl)-aminomethane
- EDTA
- ethylenediaminetetraacetic acid
- EGTA
- ethyleneglycol-bis-(β-aminoethyl ether)N,N′-tetraacetic acid
- HEPES
- (N-[2-hydroxethyl]piperazine-N′-[2-ethanesulfonic acid])
- PMSF
- phenylmethylsulfonyl flouride.
- Received September 22, 1997.
- Accepted March 9, 1998.
- The American Society for Pharmacology and Experimental Therapeutics