Introduction

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-Adrenergic receptors (ARs) are G protein-coupled receptors that are activated by the endogenous catecholamine neurotransmitters epinephrine and norepinephrine. Mutation analysis and computer modeling studies suggest that -AR activation is primarily determined by the interaction of -AR ligands with transmembrane amino acid residues (Blin et al., 1993; Granneman et al., 1998). Additional complexities in ligand-receptor interactions also play a role in defining the activity and selectivity of compounds at -AR. For example, the ₂-AR agonist salmeterol interacts with an "exo" site on the ₂-AR in addition to its interaction with amino acid residues within the transmembrane region (Green et al., 1996). Furthermore, receptors are capable of existing in an "active" or "inactive" conformation or state, and the ability of a ligand to stabilize a particular conformation of the receptor results in its classification as an agonist, antagonist, or inverse agonist (Samama et al., 1994; Kenakin et al., 1995). Recent studies also suggest that receptors can exist in multiple active conformations (Tuek, 1997; McLatchie et al., 1998; Muff et al., 1999; Seifert et al., 1999; Zuscik et al., 1998). Thus, differences in the ability of ligands to interact with and stabilize these receptor conformations/states would predict a gradation in the activities of different classes of compounds at -AR (Kenakin et al., 1995; Ganguli et al., 1998).

The aryloxypropanolamine class of adrenergic ligands displays diverse activities at -AR, where they function as agonists, inverse agonists, or antagonist. The aryloxypropanolamine CGP 12177 was initially described as a high-affinity antagonist of ₁- and ₂-ARs (Staehelin and Simons, 1982; Staehelin et al., 1983). It was later demonstrated to be a potent yet partial agonist of the rodent and human ₃-AR (Mohell and Dicker, 1989; Granneman et al., 1991, 1993; Granneman and Whitty, 1991). The relatively high potency of CGP 12177 has prompted the development of other ₃-AR-selective aryloxypropanolamines with higher efficacies, such as LY 362884 (Arch and Wilson, 1996; Jesudason, 1998). Interestingly, CGP 12177 also increases inotropic and chronotropic responses in the heart, as well as lipolysis in the adipose tissue of mice lacking ₃-AR (Kaumann et al., 1998; Preitner et al., 1998). The responses to CGP 12177 in these tissues are resistant to blockade by -AR antagonists, which is in contrast to the agonist responses induced by catecholamines such as isoproterenol. Based on this unusual pharmacological profile of CGP 12177, the presence of a fourth -AR subtype was recently proposed (Galitzky et al., 1997; Kaumann and Molenaar, 1997; Malinowska and Schlicker, 1997; Molenaar et al., 1997).

The identity of the receptor mediating the ₃-AR-independent effects of CGP 12177 described earlier was clarified in a recent study on brown fat from -AR knockout mice (Konkar et al., 2000). This study demonstrated that activation of adenylyl cyclase by CGP 12177 in ₃-AR knockout mice is mediated by the ₁-AR. Significantly, the ₁-AR-induced responses to CGP 12177 were resistant to -AR blockade, implying that they result from a novel interaction of CGP 12177 with ₁-AR. In this study, we have further characterized the pharmacological properties of the ₁-AR response to CGP 12177 and examined whether other aryloxypropanolamines that have been developed as ₃-AR agonists exhibit similar behavior. We confirm here that CGP 12177 stimulates ₁-AR and that activation of ₁-AR by CGP 12177 is resistant to blockade by -AR antagonists, in contrast to activation by catecholamines. We also show that a second aryloxypropanolamine, LY 362884, has pharmacological properties similar to CGP 12177. Our results suggest that differences in aryloxypropanolamine versus catecholamine sensitivity to -blockers are a result of their interactions with distinct active conformations or states of the ₁-AR.

	Experimental Procedures

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Materials. ()-Isoproterenol bitartrate, ()-propranolol hydrochloride, and dobutamine hydrochloride were obtained from Sigma Chemical Co. (St. Louis, MO). ()-Norepinephrine bitartrate, CGP 12177, and CGP 20712A were obtained from Research Biochemicals International (Natick, MA). LY 362884 was obtained from Eli Lilly (Indianapolis, IN). SB 251023 and SB 226552 were obtained from SmithKline Beecham Pharmaceuticals (King of Prussia, PA). The chemical structures of aryloxypropanolamine ligands used in this study are illustrated in Fig. 1. Materials for adenylyl cyclase assays were obtained from sources previously described (Chaudhry and Granneman, 1991). Ham's F-12 medium was purchased from Irvine Scientific (Santa Anna, CA). Minimum essential medium, penicillin/streptomycin, and geneticin were obtained from Life Technologies (Gaithersburg, MD). All other chemicals were of reagent grade.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Chemical structures of aryloxypropanolamine ligands used in this study.

Cell Culture. Chinese hamster ovary (CHO) and HEK 293 cells were grown in Ham's F-12 and minimum essential medium, respectively, supplemented with 10% fetal calf serum, L-glutamine (2 mM), penicillin (100,000 units/l), and streptomycin (100 mg/l). CHO and HEK 293 cells were grown at 37°C in a humidified atmosphere of 5% and 10% CO₂, respectively. Cells were harvested at approximately 90% confluence. The rat and human ₁-ARs were cloned and expressed in either CHO or HEK 293 cells as described previously (Chaudhry et al., 1992; Konkar et al., 2000). The rat ₁-AR was expressed at 43 fmol/mg of protein and 3.6 pmol/mg of protein in CHO and HEK 293 cells, respectively. The human ₁-AR was expressed in CHO cells at 40 fmol/mg of protein. The human ₁-AR cDNA was a gift from Dr. S. Liggett (University of Cincinnati, Cincinnati, OH).

Preparation of Membranes. Cells were washed twice with PBS (pH 7.4) and then lysed in a hypotonic homogenization buffer containing 25 mM HEPES (pH 8.0), 2 mM MgCl₂, 1 mM EDTA, and 10 µg/ml leupeptin. Cell membranes were collected with a rubber policeman and pelleted by centrifugation at 48,000g for 15 min at 4°C. Membranes were stored at 80°C and were resuspended in homogenization buffer immediately before use in experiments.

Adenylyl Cyclase Assay. Adenylyl cyclase activity was determined according to a modification (Granneman et al., 1991) of the method of Salomon (1979). Briefly, membranes (5-20 µg of protein) were incubated with agonists at 4°C in the presence or in the absence of various -AR antagonists for 20 min. The adenylyl cyclase reaction was initiated by the addition of the substrate mix and continued for 20 min at 30°C. The contents of the substrate mix and chromatographic separation of cAMP have been described previously (Chaudhry and Granneman, 1994). Any deviations from the methods described here are provided in legends to figures.

Statistical Analysis. Data are presented as mean ± S.E. Student's t test was used to evaluate differences between means, and critical values of P < .05 were judged statistically significant.

	Results

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Agonist Responses to Aryloxypropanolamines at ₁-AR. The aryloxypropanolamine CGP 12177 (see Fig. 1 for structure) is an agonist of ₁-AR (Fig. 2), as previously reported (Pak and Fishman, 1996; Konkar et al., 2000). CGP 12177 exhibited potent but partial agonist activity in CHO cells expressing low levels (approximately 40 fmol/mg of protein) of rat or human ₁-AR and had agonist potencies (EC₅₀ values) of 14.8 and 12.5 nM, respectively. In HEK 293 cells overexpressing (3.6 pmol/mg of protein) rat ₁-AR, CGP 12177 stimulated the rat ₁-AR with a potency of 3.3 nM (Fig. 2; Table 1). CGP 12177, however, did not stimulate adenylyl cyclase activity in untransfected cells (data not shown). Thus, activation of ₁-AR by CGP 12177 was independent of cell type and was observed for both rat and human ₁-AR. The agonist response to CGP 12177 suggested that other aryloxypropanolamine ligands developed as ₃-AR agonists might also display agonist activity at ₁-AR. Consistent with this hypothesis, we observed that the aryloxypropanolamine ligands LY 362884 and SB 251023 (Jesudason et al., 1998; Arch et al., 1999) elicited partial agonist responses at ₁-AR expressed in CHO cells. SB 226552 (Sennitt et al., 1998) stimulated ₁-AR, but only at the highest concentrations used (100 µM) (Table 1).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Comparison of CGP 12177-, isoproterenol- and norepinephrine-induced activation of rat (top and middle) and human (bottom) 1-ARs expressed in CHO and HEK 293 cells. Adenylyl cyclase activation is used as a measure of 1-AR activation. Data are presented as a percentage of the maximal response to isoproterenol. Values are means ± S.E. of 4 to 16 experiments

View this table: [in this window] [in a new window]   TABLE 1 Pharmacological parameters of rat and human 1-AR activation by catecholamine and aryloxypropanolamine ligands

Antagonism of Catecholamine-Induced ₁-AR Activation by Aryloxypropanolamine Ligands. CGP 12177 was initially described as a high-affinity ligand of the ₁- and ₂-AR that did not stimulate these receptors in tissues except at high concentrations (Staehelin et al., 1983). Our results showed that CGP 12177 stimulated ₁-AR with relatively high potency. We therefore examined whether CGP 12177 and LY 362884 behaved as high-affinity antagonists by examining their effects on isoproterenol-induced stimulation of ₁-AR. CGP 12177 (10 nM) and LY 362884 (1 nM) shifted the isoproterenol concentration-response curve rightward by 6- and 2-fold, respectively (Fig. 3). The calculated affinity estimates (K_B values) were 2 and 0.8 nM for CGP 12177 and LY 362884, respectively. Thus, both CGP 12177 and LY 362884 were potent blockers of ₁-AR. The aryloxypropanolamine SB 251023 (10 µM), however, failed to block isoproterenol-induced stimulation of ₁-AR (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Antagonism of isoproterenol-induced stimulation of rat 1-adrenergic receptors expressed in CHO cells by 10 nM CGP 12177 (top), 1 nM LY 362884 (middle), and 10 µM SB 251023 (bottom). Adenylyl cyclase activation is used as a measure of 1-AR stimulation. Values are mean ± S.E. of three or four experiments.

Antagonist and Agonist Response to Aryloxypropanolamine Compounds at ₁-AR. The agonist responses to CGP 12177 and LY 362884 at ₁-AR occurred at concentrations seven and four times greater, respectively, than those required to block the ₁-AR (Figs. 2 and 3), implying that the antagonist and agonist activities of these compounds represent distinct interactions with the ₁-AR that can be distinguished by concentration. We therefore constructed concentration-response curves to CGP 12177 and LY 362884 in the presence and in the absence of isoproterenol. A concentration of isoproterenol (10 nM) that is equieffective to the maximal response of either aryloxypropanolamine was used in these experiments. The catecholamine partial agonist dobutamine was used as a reference control.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effect of CGP 12177, LY 362884, SB 251023, and dobutamine on isoproterenol-stimulated adenylyl cyclase activity at rat 1-ARs expressed in CHO cells. CHO cell membranes expressing the rat 1-AR were stimulated for 30 min at 30°C with increasing concentrations of CGP 12177 (A), LY 362884 (B), SB 251023 (C), or dobutamine (D) in the presence or in the absence of isoproterenol. Values presented are mean ± S.E. of three experiments.

Effect of -AR Antagonists on Response to CGP 12177 and LY 362884. The observation that low concentrations of CGP 12177 and LY 362884 block ₁-AR, whereas high concentrations stimulate ₁-AR suggested that these compounds interact with distinct ₁-AR sites or states to elicit antagonist and agonist responses. These results also suggest that the catecholamine-responsive site or state is distinct from the aryloxypropanolamine-responsive site or state. Thus, we hypothesized that -blockers might differentially block these distinct sites or states of the receptor. In the following experiments, we compared the relative potencies of the -AR antagonists propranolol and CGP 20712A for blocking activation of ₁-AR by catecholamines and aryloxypropanolamines.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Antagonism of isoproterenol- and CGP 12177-induced activation of human 1-ARs by increasing concentrations of CGP 20712A in CHO cells (top and bottom, respectively). Adenylyl cyclase activation is expressed as a percentage of the maximal response to isoproterenol or CGP 12177. Insets, corresponding Schild plots. Values are presented as mean ± S.E. of four to six experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Antagonism of isoproterenol-, norepinephrine-, and CGP 12177-induced activation of human 1-ARs by increasing concentrations of propranolol in CHO cells (top, middle, and bottom, respectively). Adenylyl cyclase activation is expressed as a percentage of the maximal response to isoproterenol, norepinephrine, or CGP 12177. Insets, corresponding Schild plots. Values are presented as mean ± S.E. of three to seven experiments.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

CGP 12177 was initially described as a high-affinity antagonist of ₁- and ₂-ARs that possessed moderate sympathomimetic properties at higher concentrations (Staehelin et al., 1983). The compound was later observed to be a potent but partial agonist of the rodent and human ₃-AR (Mohell and Dicker, 1989; Granneman et al., 1991, 1993) and was used as a prototypical drug for developing highly efficacious ₃-AR selective agonists (Arch and Wilson, 1996). In recent studies, CGP 12177 was observed to stimulate cardiovascular and metabolic responses that were resistant to -AR blockers in wild-type and ₃-AR knockout mice (Kaumann et al., 1998; Preitner et al., 1998). These findings suggested the presence of a novel -AR subtype that has been designated ₄-AR (Galitzky et al., 1997; Kaumann and Molenaar, 1997). A more recent study demonstrated that CGP 12177-induced responses attributed to activation of the putative ₄-AR in native tissues actually occur through its interaction with ₁-AR (Konkar et al., 2000). We have therefore used pharmacological analyses to examine in detail the interaction of CGP 12177 with ₁-AR in a cell expression system. We have also examined whether other aryloxypropanolamines, developed as ₃-AR agonists (Jesudason, 1998; Sennitt et al., 1998; Arch et al., 1999), exhibit behavior similar to that of CGP 12177 at ₁-AR.

In concurrence with earlier observations, low concentrations of CGP 12177 potently blocked isoproterenol-induced activation of ₁-AR (Staehelin et al., 1983), whereas at higher concentrations, CGP 12177 behaved as an agonist. Another aryloxypropanolamine, LY 362884, displayed behavior similar to that of CGP 12177. Independent estimates showed that inhibitory potencies (K_B values) of CGP 12177 and LY 362884 were 7- and 4-fold higher than their stimulatory potencies (EC₅₀ values), respectively. Thus, in the presence of an equieffective concentration of isoproterenol, both CGP 12177 and LY 362884 exhibited a biphasic response at the ₁-AR. At low concentrations, CGP 12177 and LY 362884 blocked ₁-AR stimulation by isoproterenol, whereas higher concentrations of these compounds stimulated the ₁-AR. The ₁-AR stimulation profiles of these compounds in the presence of isoproterenol were identical to the profiles obtained in the absence of isoproterenol. This behavior suggests that although both aryloxypropanolamines block activation of ₁-AR by isoproterenol, they also stimulate ₁-AR independently of the catecholamine-responsive site or state. The biphasic effect demonstrated by CGP 12177 and LY 362884 is in stark contrast to that observed for the catecholamine dobutamine and the aryloxypropanolamine SB 251023. The latter two compounds exhibit a monotonic inhibition of isoproterenol-stimulated adenylyl cyclase activity, as predicted for simple partial agonists (Ariëns, 1983). Another aryloxypropanolamine (SB 226552) evaluated in this study produced a weak stimulation of the ₁-AR and consequently could not be used for detailed pharmacological analyses. However, an earlier study shows that this compound does not interact with the putative ₄-AR (Sennitt et al., 1998) and thus would be expected to behave in a manner analogous to dobutamine and SB 251023. These data suggest that the partial agonists dobutamine and SB 251023 interact with the catecholamine-responsive site or state of ₁-AR. In contrast, the aryloxypropanolamines CGP 12177 and LY 362884 appear to interact with two different sites or states of the ₁-AR.

The presence of an aryloxypropanolamine-responsive ₁-AR site or state that is distinct from the catecholamine-responsive state raises the possibility that these two sites or conformations may differ in their interactions with -AR ligands. We therefore examined the ability of -AR antagonists to block responses to aryloxypropanolamines and catecholamines to further characterize the aryloxypropanolamine-responsive state of ₁-AR. If aryloxypropanolamines and catecholamines interact with the same site or conformation of ₁-AR to produce agonist responses, -AR antagonists should block responses to each compound with equal potency. However, we observe that CGP 12177- and LY 362884-induced responses at ₁-AR are consistently and significantly resistant to -AR blockade compared with the responses to the catecholamines isoproterenol, norepinephrine, and dobutamine. These data provide further evidence indicating that catecholamines and aryloxypropanolamines interact with distinct active conformations of ₁-AR.

In this study, we used CGP 12177 in functional assays to define a novel state of ₁-AR. It is unclear, however, whether this novel state can be labeled and defined using currently available radioligands. In earlier studies, Pak and Fishman (1996) demonstrated that CGP 12177 binds to a high- and a low-affinity site of ₁-AR. The low-affinity CGP 12177 binding site constituted ~10% of the receptor population and was sensitive to the guanine nucleotide guanosine-5'-O-(3-thio)triphosphate, indicating that it is the agonist-binding site for CGP 12177. In native tissues, however, [³H]CGP 12177 labels a site that is insensitive to propranolol blockade and guanosine-5'-O-(3-thio)triphosphate, and this site was tentatively defined as the putative ₄-AR (Kaumann et al., 1998; Sarsero et al., 1998, 1999). Studies using ₁- and ₃-AR knockout mice show that ₁-AR activation completely accounts for the ₄-AR-like profile seen in native tissues (Rohrer et al., 1996; Konkar et al., 2000). It is thus likely that the propranolol-resistant [³H]CGP 12177 binding observed in native tissues represents an interaction between CGP 12177 and the novel state of the ₁-AR defined here. Our study shows that CGP 12177 activity at ₁-AR is resistant to blockade by propranolol. However, propranolol discriminates between the two active states of ₁-AR with rather low selectivity. Thus, this antagonist seems unsuitable to accurately define the novel ₁-AR state in radioligand binding studies. Further characterization will be possible with the development of highly selective ligands for the aryloxypropanolamine-sensitive state of the ₁-AR.

Receptors are thought to exist in an active state or an inactive state, and their ligands are classified as agonists, inverse agonists, or neutral antagonists based on their interactions with these states. Agonists preferentially stabilize the active state, whereas inverse agonists stabilize and promote the inactive state of the receptor. Neutral antagonists, however, lack preference for either the active or the inactive receptor state (Kenakin et al., 1995). These results indicate that CGP 12177 and LY 362884 do not fall into one or the other category but possess both antagonist and agonist properties at ₁-AR that are concentration-dependent. Molecular modeling indicates that -AR ligands can assume either folded or extended conformations, and only the latter conformation allows receptor activation (Blin et al., 1993). The relatively constrained ligand binding pockets of ₁- and ₂-AR allow interaction with the folded conformations of -AR ligands, where they behave as antagonists. The less constrained binding pocket of the ₃-AR accommodates extended conformations of -AR ligands and results in receptor activation. Significantly, ₃-AR activation by catecholamines and aryloxypropanolamines is poorly blocked by propranolol. Thus, the novel ₁-AR state activated by aryloxypropanolamines seems to possess pharmacological properties similar to the ₃-AR. The two ₁-AR active states described here may allow binding of folded and extended conformations, with the aryloxypropanolamine-responsive state exhibiting ₃-AR-like properties of activation by extended conformers and relatively low affinity for propranolol.

Pak and Fishman (1996) found a direct correlation between ₁-AR receptor number and CGP 12177 responsiveness in a heterologous expression system. Interestingly, the proportion of the CGP 12177 responsive sites, however, remained constant regardless of the change in expression level of ₁-AR. Although these results agree with earlier analysis correlating CGP 12177 responsiveness to receptor expression, overexpression of ₁-AR does not appear to be a prerequisite for CGP 12177 response because agonist activity was observed in native tissues (Konkar et al., 2000) and CHO cells expressing rat and human ₁-AR at physiological levels. Moreover, expression level does not alter the antagonist potencies because CGP 20712 blocked the rat ₁-AR with similar potencies when expressed at physiological levels or at a level approximately 100-fold greater. The molecular basis of the atypical state of the ₁-AR is unknown. The CGP 12177-sensitive state might be generated through posttranslational modifications of the receptor or represent homomeric or heteromeric protein interactions. In this regard, we have noted that the activity of CGP 12177 relative to isoproterenol varies among tissues (our unpublished observations), suggesting that the stabilization of the atypical ₁-AR state may involve interactions with other proteins (Tang et al., 1999), perhaps in a manner analogous to that described for calcitonin gene-related peptide receptors and receptor activity modifying proteins (McLatchie et al., 1998). Clearly, further work is needed to clarify the molecular basis of aryloxypropanolamine activation of ₁-AR and the impact of these ligands on ₁-AR signaling.

These results also have implications for the use of antagonist affinity values (pA₂ or K_B values) in defining and classifying receptors. Antagonists affinity values are used to define receptor subtypes because classic receptor theory maintains that the potency of an antagonist for a given receptor subtype remains constant regardless of the agonist used to elicit the response (Kenakin, 1982; Kenakin et al., 1992). Current receptor theory models also indicate that a given receptor can exist in a conformation that is either active or inactive (Samama et al., 1994). Recent studies suggest that a given receptor can exist in more than one active conformation (Tuek, 1997; McLatchie et al., 1998; Muff et al., 1999; Seifert et al., 1999; Zuscik et al., 1998). Agonist ligands can thus be predicted to interact with receptor-binding sites differentially based on the conformation presented (Ganguli et al., 1998), and hence antagonist affinities at a given receptor might differ in an agonist-dependent fashion. Consistent with this hypothesis and in contrast to current receptor theory models, we find that antagonist affinities at ₁-AR can be agonist-dependent.

In summary, this report presents evidence indicating that the aryloxypropanolamine CGP 12177 is an antagonist of ₁-AR at low concentrations and an agonist of ₁-AR at high concentrations. In contrast to catecholamines, the agonist response to CGP 12177 is resistant to -AR blockade. Another aryloxypropanolamine, LY 362884, also displays an unusual pharmacological profile similar to that exhibited by CGP 12177. The blockade of catecholamine-induced stimulation of ₁-AR by CGP 12177 and LY 362884 and the resistance of CGP 12177- and LY 362884-induced activation of ₁-AR to -blockers suggest that certain aryloxypropanolamine compounds can interact with at least two distinct active conformations of ₁-AR.