Introduction

Top Abstract Introduction Materials and Methods Results Discussion Appendix References

Receptors coupled to G proteins (GPCRs) play a major role in signal transduction and are the targets of a large number of therapeutic drugs. An important development in the understanding of GPCRs was the recognition that they could couple to (Wreggett and De Lean, 1984; Neubig et al., 1988) and functionally activate (Costa and Herz, 1989) G proteins in the absence of an agonist. These initial observations were buttressed by the identification of mutant receptors that had substantial constitutive activity (Kjelsberg et al., 1992; Parma et al., 1993; Shenker et al., 1993). This led to the "Extended Ternary Complex (ETC) model" (Samama et al., 1993) and the cubic ternary complex model (Weiss et al., 1996). In these models, the receptor exists in an equilibrium between an inactive state R and an active state R* in the absence of drug. This equilibrium, differing for each receptor, determines basal activity. For wild-type receptors, R predominates and there is minimal receptor activity in the absence of agonist. Binding of agonist stabilizes R* causing G protein coupling and activation of cellular responses. High levels of receptor expression or constitutively active mutant receptors increase the concentration of R* inducing a response in the absence of agonist. In some disease states such as familial male precocious puberty (luteinizing hormone receptor) (Shenker et al., 1993), hyperfunctioning thyroid adenoma (thyrotropin receptor) (Parma et al., 1993), and retinitis pigmentosa (rhodopsin) (Rao et al., 1994), naturally occurring point mutations that lead to constitutive activation of GPCRs have been implicated.

Constitutively active GPCRs suggested the concept of inverse agonists (Barker et al., 1994; Chidiac et al., 1994; Bond et al., 1995)drugs that preferentially bind to R and inhibit basal receptor activity. In contrast, neutral antagonists bind equally well to R and R*, have no effect on basal receptor activity, but block the effects of both agonists and inverse agonists. Indeed, many drugs classically thought of as antagonists were subsequently found to have inverse agonist activity (Chidiac et al., 1994). Inverse agonists are expected to have therapeutic utility in the setting of CAM receptors such as in familial male precocious puberty or thyroid adenomas but more recently, they have been found to have unique physiological or biochemical effects (Nagaraja et al., 1999) even in the setting of relatively low levels of expression of wild-type receptors (Berg et al., 1999). Thus the differentiation of inverse agonist and neutral antagonist activity is important to our understanding of drug mechanisms.

Although the general features of the ETC model are accepted, there have not been clear quantitative tests of that model. For example, it isn't clear what fraction of receptors are in the R* state for WT and CAM receptors. Also, inverse agonists should have a lower affinity for the R* state of GPCRs but the data demonstrating this is not clear. Most studies have shown limited (2-fold) effects on inverse agonist affinity for CAM mutant receptors. Here we examine quantitative questions about the ETC model in the context of the _2A-adrenergic receptor.

₂-Adrenergic receptors (AR) play important physiological roles in the central control of blood pressure, pain, and neuronal excitability and the _2A-AR subtype is the major contributor to these responses (MacMillan et al., 1996; Hein et al., 1999). Furthermore, ₂-AR agonists like clonidine are effective antihypertensive therapies although there remains some controversy over the relative role of the _2A-AR and the putative I₁ imidazoline receptor in these actions. Like opioid receptors, ₂-ARs are well known to exhibit a pronounced excitatory withdrawal syndrome upon rapid cessation of chronic agonist therapy (Neusy and Lowenstein, 1989). The classical antagonist of the ₂-AR, yohimbine, causes central excitation but it is not clear whether the central excitatory effects of yohimbine are: 1) manifestations of pure antagonist activity at the ₂-AR, 2) due to the reported inverse efficacy of yohimbine (Tian et al., 1994), or 3) due to effects on other receptors such as 5HT receptors (Convents et al., 1989). Currently, there is insufficient pharmacological characterization of these _2A antagonists to define such mechanisms. The identification of _2A adrenergic inverse agonists and neutral antagonists that are more selective than yohimbine would be desirable. Furthermore, recent literature is contradictory as to whether the classical ₂-AR antagonists are inverse agonists or neutral antagonists (Tian et al., 1994; Wurch et al., 1999).

Lefkowitz and colleagues (Ren et al., 1993) showed that mutation of Thr-373 in the _2A-AR results in CAM activity leading to basal inhibition of adenylyl cyclase and increased receptor affinity for agonist. We use this CAM _2A-AR to evaluate ₂-antagonists for inverse agonist activity. We wished to determine inverse efficacy of ₂-AR antagonists and to examine quantitative predictions of the ETC model. We found inverse agonist activity among both alkaloid and imidazoline ₂-antagonists and identified a unique difference between idazoxan and RX821002, which may have important pharmacological implications. Also, we obtain estimates of the fraction of the _2A-T373K mutant receptor in the R* state and set an upper bound for that fraction for the WT _2A-AR.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Appendix References

Radiochemicals. [2-³H]Adenine (21-25 Ci/mmol) was from Amersham Life Science (Arlington Heights, IL). [³H]Yohimbine (74.5-78 Ci/mmol) was from DuPont-New England Nuclear (Wilmington, DE).

Chemicals. Opti-MEM, LipofectAMINE, and Geneticin (G-418) were obtained from Life Technologies (Gaithersburg, MD). Fluorescein-labeled anti-[HA]-antibody was from Boehringer Mannheim (Indianapolis, IN). Pertussis toxin (PTX) and cholera toxin were from List Biological Laboratories (Campbell, CA). Forskolin was from Calbiochem (La Jolla, CA). UK-14,304 and prazosin were from Pfizer (Sandwich, England). Clonidine was from Boehringer Ingelheim (Ingelheim, Germany), idazoxan from Reckitt & Colman (Hull, England), MK912 a gift of Dr. Staffan Uhlén, Uppsala University, phenoxybenzamine from Smith Kline & French Labs (Philadelphia, PA), phentolamine from Ciba-Geigy (Summit, NJ), propranolol from Ayerst Laboratories (New York, NY), rauwolscine from Roth (Germany), and RX821002 from Research Biochemicals, Inc. (Natick, MA). Isobutyl-1-methyl-xanthine (IBMX), adenosine 5'-triphosphate (ATP), adenosine 3':5'-cyclic monophosphate (cAMP), 5'-guanylyimidodiphophate (GppNHp), epinephrine and yohimbine were from Sigma (St. Louis, MO).

Construction of Mutant _2A-Adrenergic Receptor Plasmids. The p₂Tag H/N construct was described previously (Wade et al., 1999). It includes an HA-epitope-tagged porcine _2A-adrenergic receptor with unique silent HindIII and NheI restriction sites at Ala-359 and Lys-376. Mutagenic cassette ligation was used to introduce annealed 52-mer oligonucleotides containing the Thr-373 to lysine constitutively activating mutation into the HindIII/NheI digested p₂Tag H/N vector. The mutation of the modified region in the product was confirmed by restriction enzyme digestion utilizing a silent diagnostic NruI restriction site and confirmed by DNA sequencing.

Cell Culture and Transfection. CHO-K1 cells were maintained in Ham's F-12 medium with 10% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO₂. Stable selection for mutants was maintained by the addition of 0.4 mg/ml active G-418.

CHO-K1 Membranes. Membranes were prepared as previously described (Wade et al., 1999). The final membrane pellets were resuspended in TME buffer (50 mM Tris, 10 mM MgCl₂, 1 mM EGTA, pH 7.6), snap frozen, and stored at 80°C. Protein was determined by Bradford protein assay (Bradford, 1976).

Radioligand Binding Assays. [³H]Yohimbine binding assays were performed in 96-well plates with 2 to 5 µg of protein per well in a final volume of 100 µl as previously described (Neubig et al., 1985). For competition binding measurements, membranes were incubated with the indicated drugs in TME buffer in the presence of 10 nM [³H]yohimbine at room temperature for 30 min and filtered using a Brandel cell harvester. Nonspecific binding was defined by 10 µM yohimbine.

Whole Cell cAMP Accumulation. Whole cell cAMP accumulation was determined in 24-well plates as previously described (Wade et al., 1999). Briefly, cells were plated with 1 µCi/well [³H]adenine for 18 to 20 h before assay and where indicated, 100 ng/ml pertussis toxin or 5 µg/ml cholera toxin was included in this preincubation. Cells were washed once with DMEM, then the assay was initiated by adding DMEM containing 1 mM IBMX, 30 µM forskolin, and the indicated drugs. Cells were incubated 30 min at 37°C, and the reaction was terminated by aspirating the incubation medium and quenching with 1 ml 5% TCA containing 1 mM ATP and 1 mM cAMP. Acid soluble nucleotides were separated on Dowex and alumina columns as described by Salomon et al. (1974). cAMP accumulation was normalized by dividing the [³H]cAMP counts by the total [³H]nucleotide counts (sum of ATP and ADP counts from the Dowex columns and cAMP counts from the alumina columns).

Data Analysis. All data are reported as mean ± S.E.M. Fitted curves were determined using an unweighted nonlinear least-squares method in GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA, www.graphpad.com). Statistical analysis of K_d ratios for WT and CAM receptors used the two-tailed, one sample t test in GraphPad Prism to compare the experimental ratio with a theoretical value of 1.0.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Appendix References

Radioligand Binding and Receptor Conformation. It is well recognized that receptor conformation states can be monitored by radioligand binding. Thus we examined quantitatively both agonist and antagonist affinities for the WT and CAM _2A-AR. Saturation binding experiments with the radiolabeled ₂-antagonist [³H]yohimbine in membranes from the WT-Tag19 and CAM-C16 cell lines revealed similar levels of receptor expression with B_max values of 25 and 19 pmol/mg of protein, respectively (Table 1 and Fig. 1). Mock-transfected (Neo) cells displayed negligible specific binding (data not shown). To assess the conformational equilibria of the receptor independent of G protein coupling, we measured UK-14,304 competition for [³H]yohimbine binding in the presence of GppNHp. As expected, the CAM receptor membranes had a much higher affinity for the ₂-AR agonist UK-14,304 than did the WT receptor-containing membranes (Table 1). Three pieces of evidence indicate that this enhanced affinity (13 nM versus 92 nM) reflects receptor conformations and not G protein coupling. First, the binding was conducted in the presence of GppNHp, which should uncouple RG complex. Second, two-site fits (not shown) of the competition studies even in the absence of GppNHp revealed only a small percentage of the receptors (<20%) in the high-affinity agonist binding component and F-tests did not show a statistical improvement by two-site fits over one-site fits (p > 0.5). Third, direct [³H]UK-14,304 binding studies showed only 2 to 3 pmol/mg of binding sites versus ~20 pmol/mg of [³H]yohimbine binding sites (not shown). Thus, due to the high receptor expression levels in these stable cell lines (i.e., greater than the amount of G protein), most of the receptor is uncoupled from G protein and the increased affinity of the CAM receptor for UK-14,304 is due to the intrinsic conformational properties of the receptor rather than to altered G protein coupling.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Yohimbine saturation binding of WT and CAM porcine 2A-AR. Saturation binding to WT-Tag19 () or CAM () membranes was performed in 96-well plates using 1 to 40 nM [3H]yohimbine for 30 min at room temperature. Nonspecific binding, defined in the presence of 10 µM yohimbine, represented less than 10% of the total binding and was subtracted. Data represent the mean ± S.E.M. of three separate experiments performed in duplicate. The Scatchard plot of the saturation binding data is shown in the bottom panel.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Binding of selected agonists and antagonists at the WT and CAM 2A-AR. Competition binding for 10 nM [3H]yohimbine in the presence of 10 µM GppNHp was performed for 30 min at room temperature with WT-L1 or WT-L9 () and CAM () membranes. The agonists epinephrine and clonidine are shown in panels A and B, respectively, whereas panels C and D depict the antagonists rauwolscine and idazoxan. Data represent the mean ± S.E.M. of two (rauwolscine), three (epinephrine and clonidine), or four to five (idazoxan) separate experiments performed in duplicate. The rauwolscine data represents two experiments using the WT-L1 membranes; five additional experiments using the WT-L9 membranes gave similar results.

CAM Receptor Constitutively Inhibits cAMP Accumulation. To evaluate the functional activity of the CAM receptor and the effects of potential inverse agonists, whole cell cAMP measurements were done. Forskolin-stimulated [³H]cAMP production in CAM cells was about 20 to 25% of that seen in either mock-transfected (Neo) cells or in cells expressing the WT receptor, p < 0.01 for CAM versus Neo (Fig. 3, top). The cAMP level in cells with the CAM receptor was similar to that observed with the WT receptor in the presence of the full agonist UK-14,304. Pretreatment with pertussis toxin decreased the cAMP production in our Neo cells by about 60%, and a similar effect was seen with WT receptor expressing cells. In contrast, cAMP actually increased 3-fold in the CAM cells upon PTX treatment, abolishing the differences among the three cell types (Fig. 3, bottom, open bars). This indicates that the low cAMP production from the constitutive activity of the mutant was mediated via agonist-independent activation of G_i and that WT receptor has minimal constitutive activity on its own, despite the high-expression level (25 pmol/mg of protein). When the agonist UK-14,304 was added to WT receptor-containing cells in the absence of PTX, cAMP production was inhibited, however cAMP was stimulated by UK-14,304 in pertussis-treated WT cells. This is consistent with previous reports of dual coupling of _2A-AR to both G_i and G_s (Eason et al., 1992; Wade et al., 1999). In CAM cells, however, UK-14,304 stimulated cAMP production both with and without pertussis pretreatment (Fig. 3). This suggests that the G_i response is already fully saturated in CAM cells before the agonist is added so only a G_s response is observed. UK-14,304 had no effect on adenylyl cyclase activity in mock-transfected (Neo) cells (Brink et al., 2000).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Pertussis-sensitivity of transfected cell lines in the presence or absence of agonist. Whole cell adenylyl cyclase assays were performed on mock-transfected (Neo), WT, and CAM cells with (bottom panel) or without (top panel) PTX pretreatment. Cells were incubated with (filled bars) or without (open bars) 10 nM UK-14,304 for 30 min at 37°C in the presence of 1 mM IBMX and 30 µM forskolin. Results are the mean ± S.E.M. of three separate experiments performed in duplicate. Forskolin-stimulated [3H]cAMP production in CAM cells was about 20 to 25% of that seen in Neo cells. Values differing significantly from control (Neo without UK-14,304) are marked, p < 0.05 (*), p < 0.01 (**).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Agonist-mediated cyclase activity in WT and CAM cells. Whole cell adenylyl cyclase assays were performed on WT (top panel) or CAM (bottom panel) cells that had been pretreated with () or without () PTX or with cholera toxin (). Cells were incubated with increasing concentrations of epinephrine for 30 min at 37°C in the presence of 1 mM IBMX and 30 µM forskolin. Data are the mean ± S.E.M. of two, three (WT + PTX), or four (CAM control) separate experiments performed in duplicate or triplicate.

Inverse Efficacies of ₂-Adrenergic Antagonists. We next looked at the effects of antagonists on forskolin-stimulated cAMP accumulation in CAM cells to determine whether inverse agonist activity would be observed. Rauwolscine was reported by Tian et al. (1994) to have the greatest inverse agonist activity at [³⁵S]GTPS binding in PC12 cells. Consistent with that, we saw a striking reversal of the CAM receptor-suppressed cAMP production by that drug. Cholera toxin pretreatment of CAM cells did not affect the rauwolscine-mediated increase in cAMP accumulation (data not shown), but the increase was completely abolished by PTX pretreatment (Fig. 5), evidence of the G_i dependence of this response. Indeed there is a small decrease in cAMP with rauwolscine in PTX-treated CAM cells suggesting a small degree of constitutive activation of G_s. The WT receptor showed strikingly different results. In contrast to the constitutive activity reported for the WT rodent _2A-AR in PC12 cells (Tian et al., 1994), we saw no increase in cAMP with rauwolscine with the WT porcine _2A-AR in our CHO cells (Fig. 5, open squares). Thus despite very high levels of receptor expression (25 pmol/mg of protein) there was no evidence with either rauwolscine (Fig. 5) or PTX (Fig. 3) for constitutive activity of the WT receptor. Four of the five competitive antagonists tested showed inverse agonist activity in which the CAM receptor-suppressed cAMP production was significantly increased (Fig. 5, bottom). The sole neutral antagonist was idazoxan. That these were inverse agonist effects and not agonist effects is evident since the increase in cAMP was mediated by a PTX-sensitive G_i-dependent mechanism like that of rauwolscine rather than a G_s-dependent mechanism like epinephrine or UK-14,304 (Fig. 5, bottom).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Pertussis-sensitivity of inverse agonists in CAM cells. Whole cell adenylyl cyclase assays were performed on cells for 30 min at 37°C in the presence of 1 mM IBMX and 30 µM forskolin. Top panel, CAM cells pretreated with () or without () PTX and untreated WT cells () were incubated with increasing concentrations of rauwolscine. CAM data are the mean ± S.E.M. of two (PTX treated) or seven (control) separate experiments; WT data are from three separate experiments. Bottom panel, CAM cells that had been pretreated with (filled bars) or without (open bars) PTX were incubated with 10 µM of the indicated drugs. Data are the mean ± S.E.M. of three to six separate experiments for untreated cells and one or two separate experiments for PTX-treated cells. All experiments were performed in duplicate.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Range of inverse efficacy of 2 antagonists. Whole cell adenylyl cyclase assays on CAM cells were carried out for 30 min at 37°C in the presence of 1 mM IBMX, 30 µM forskolin, and increasing concentrations of the indicated drugs. Data are the mean ± S.E.M. of three to seven separate experiments performed in duplicate.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Appendix References

We have used a constitutively active mutant of the _2A-AR stably expressed in CHO-K1 cells to examine mechanisms of _2A-AR signaling. From these studies, at least three new conclusions can be drawn. First, the _2A-AR T373K has enhanced signaling to both G_i and G_s. Second, the inverse efficacy of a series of ₂-AR antagonists is examined, and a novel difference between idazoxan and methyl-idazoxan (RX821002) has been found. This point is important for interpreting physiological studies that use these two compounds. Finally, the fraction of active _2A-AR for the T373K CAM mutant is approximately 50% for our whole cell cAMP accumulation assay, whereas the WT receptor appears to have a very low percentage of receptor active in the absence of agonist.

The effects of constitutively active receptor mutants on different G proteins are not always concordant as reported for the ₁-AR by Perez et al. (1996). Ren et al. (1993) previously showed for the _2A-AR that the T373K mutation adjacent to TM6 causes dramatically enhanced basal activation of G_i, a result that we confirm here. We also demonstrate enhanced activation of G_s by the T373K mutant. This appeared as a modest degree of basal G_s response with the T373K mutant (Fig. 5, filled circles), but a more pronounced effect was seen on the EC₅₀ for epinephrine-induced G_s activation. The EC₅₀ was reduced >50-fold for the mutant versus WT whereas the K_d was reduced less than 10-fold, indicating enhanced G_s coupling. This does not appear to be just a shift in activity for G_i versus G_s since G_i effects were eliminated in this experiment by PTX treatment. However, this is difficult to evaluate quantitatively in part due to the fact that the _2A-AR has a much reduced ability to activate G_s compared with G_i (Eason et al., 1992; Brink et al., 2000). Wurch et al. (1999) found increased G_z coupling by the T373K mutant in a transient cotransfection system with mouse G₁₅ in COS-7 cells using inositol phosphate production as their readout. Thus, the T373K mutant of the _2A-AR appears to have a generalized ability to activate both G_i and G_s although quantitatively a comparison of the degree of activation is not possible.

Interestingly, the pharmacological behavior of this basal G_z activation was distinct from our results. For adenylyl cyclase inhibition, we show a clear order of inverse efficacy of rauwolscine > yohimbine > RX821002 > MK912 with RX821002 having substantial inverse efficacy. Phentolamine and idazoxan were neutral antagonists. In contrast, Wurch et al. (1999) reported that the basal G_z-mediated PLC activation with the T373K _2A-AR could not be attenuated by RX821002, MK912 or idazoxan. They did not, however, report results with the most efficacious inverse agonists, rauwolscine or yohimbine. We would have expected a significant reduction in activation of G_z by RX821002 if it behaved in a manner similar to what we observe for G_i. Studying the rat _2D-receptor (which is an ortholog of the human and porcine _2A-AR), Tian et al. (1994) used [³⁵S]GTPS binding in PC12 cells to show that yohimbine, rauwolscine, phentolamine, and idazoxan were all inverse agonists. They reduced basal GTPS binding by 30 to 40% but that group did not test RX821002 or MK912. Interestingly, their ability to detect constitutive signaling (and inverse agonist effects) was abolished when sodium chloride was included in the assay.

For the endogenous _2C-receptor in HepG2 cells, Cayla et al. (1999) found that treatment with RX821002 or yohimbine resulted in a significant increase in receptor number but phentolamine did not. They concluded that RX821002 and yohimbine thus behaved as inverse agonists, whereas phentolamine behaved as a neutral antagonist in this system. Although this was a different receptor subtype and a different experimental readout, their pharmacological profile completely agrees with ours. Interestingly, we were not able to demonstrate any significant changes in _2A-receptor number after treatment of either WT or CAM _2A-AR with UK-14,304 or rauwolscine (data not shown). This difference may be due to the well known resistance of _2A-AR to internalization and the substantial pool of _2C-AR, which is intracellular in the absence of agonist (Daunt et al., 1997).

Thus several factors, such as the type of assay, receptor subtypes, cell type, and local cellular G protein concentrations may affect constitutive receptor activity and thus will be important for detection of inverse agonist activity at a receptor subtype (for review, see Pauwels and Wurch, 1998). Interestingly, the best concordance of the pharmacological data mentioned above is between our results with _2A-AR-mediated inhibition of cAMP accumulation and the receptor up-regulation studies of Cayla et al. (1999) with _2C-AR. Yohimbine and RX821002 were inverse agonists in both studies. There is a direct contradiction between our results and those of Tian et al. (1994) in that phentolamine and idazoxan are clearly neutral antagonists in our hands. In particular, idazoxan showed both neutral antagonist behavior in the functional assay and absolutely no preference for R* in the binding studies.

One of the other major questions that we address here is the fraction of receptor that is active in the absence of agonist. Also, we wanted to understand why CAM mutations have minimal effect on apparent affinity of even the most efficacious inverse agonists. The two-state ETC model has been frequently used to explain constitutive activation and inverse agonists (Samama et al., 1993), and it would predict that an inverse agonist must have a substantial difference in affinities for R and R*. Although some recent data indicate that either an induced fit model or multiple active receptor states may be required to account for observations such as agonist-directed trafficking of receptor stimulus (Kenakin, 1995) or differential constitutive activation of signaling pathways (Perez et al., 1996), the ETC model is still a useful starting point for analysis. Although a lower affinity of inverse agonists for CAM receptors has been observed (Costa and Herz, 1989; Samama et al., 1994), the effects were very small (2-fold), and numerous other studies have not reported such an effect (Kjelsberg et al., 1992; Ren et al., 1993). In our theoretical examination of this question (see Appendix), we show that the expected changes in antagonist affinity are indeed modest and depend on the degree to which the receptor is spontaneously in the active state (i.e., depends on L, the equilibrium constant governing the R to R* equilibrium). Only if a very large fraction of the receptor (i.e., >90%) is spontaneously in the R* state would you expect to see a large affinity shift of an inverse agonist for a WT versus CAM receptor. The approximately 2-fold shift that we see for rauwolscine is consistent with approximately half of the CAM receptor being in the R* state (see Appendix for details). Furthermore, the lack of inverse agonist effect with the WT _2A-AR despite very high expression indicates that a very small percentage of the WT receptor is active in the absence of agonist. This is in contrast to the ₂-AR for which the WT receptor appears to have approximately 7% of the activity of the CAM mutant (Samama et al., 1993).

This very low level of constitutive activity of the WT _2A-AR seems in conflict with results of Tian et al. (1994). They used PC-12 cells stably expressing a cloned rat _2D-AR to look at the effects of rauwolscine on [³⁵S]GTPS binding to membrane preparations. The _2D-AR is the rodent ortholog of the human or porcine _2A-AR. They found that rauwolscine was able to decrease basal [³⁵S]GTPS binding and that this decrease was dependent on both the level of receptor expression and upon sodium concentration in their assay (as noted above). Furthermore, their pharmacological profile of inverse efficacy was very different from ours. There are many potential explanations for the differences between our results and theirs: 1) the different species of origin of the receptor (rat versus pig), 2) different cell types used (CHO versus PC12), 3) the different readout of the assay (GTPS binding versus adenylyl cyclase inhibition), and 4) the presence of sodium in our intact cell cAMP accumulation assays, which was absent from their nucleotide binding studies. Sodium, which is present in intact systems, has been shown to suppress basal activity for opioid receptors (Costa et al., 1990).

A final observation relates to different inverse efficacies of closely related imidazoline compounds. A role has been postulated for a novel imidazoline I₁ type receptor in mediating many effects of clonidine and other imidazoline-containing ₂ agonists (Ernsberger and Haxhiu, 1997). One pharmacological distinction between imidazoline receptor effects and those of ₂-AR has been the difference between effects of idazoxan and its methyl-substituted congener RX821002. This difference has been ascribed to selectivity of RX821002 to bind to the ₂-AR but not to the putative I₁ receptor. In contrast, idazoxan binds to both types of sites (Meana et al., 1997). Interestingly in our study, RX821002 (2-methoxy idazoxan) is a fairly efficacious inverse agonist whereas idazoxan is a neutral antagonist. Although different types of evidence have pointed to the I₁ receptor as the mediator of imidazoline drug-induced physiological effects, differences between the actions of RX821002 and idazoxan or other compounds must now take into account the relative inverse efficacy of these drugs at the _2A-AR.

In conclusion, we describe the relative inverse efficacies of a series of _2A-AR antagonists and the relation of inverse efficacies to binding properties. We provide new information both about _2A-adrenergic receptor conformational regulation as well as potentially important pharmacological distinctions among different _2A-AR antagonists.