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CELLULAR AND MOLECULAR

Evidence for Pleiotropic Signaling at the Mouse ₃-Adrenoceptor Revealed by SR59230A [3-(2-Ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol Oxalate]

Department of Pharmacology, Monash University, Victoria, Australia (D.S.H., M.S., B.A.E., R.J.S.); and Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia (A.C.)

Received for publication August 30, 2004
Accepted November 30, 2004.

	Abstract

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This study examines the action of the ₃-adrenoceptor antagonist SR59230A [3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanoloxalate] at cloned mouse ₃-adrenoceptors expressed in Chinese hamster ovary cells (CHO-K1-₃) or endogenously expressed in 3T3-F442A adipocytes or ileum. SR59230A displayed partial agonist properties compared with the ₃-adrenoceptor agonist CL316243 [GenBank] [(R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate] in CHO-K1-₃ with the intrinsic activity increasing with the level of receptor expression. Functional affinity values for SR59230A at each level of receptor expression were in agreement with pK_I values determined by binding. In cytosensor microphysiometer studies, SR59230A was a full agonist for increases in extracellular acidification rates (ECARs) at all levels of receptor expression, and antagonist actions were measurable only in medium- or low-expressing cells. In 3T3-F442A adipocytes, SR59230A antagonized CL316243 [GenBank] -mediated increases of cAMP and had no agonist actions. However, in the cytosensor micro-physiometer, SR59230A (acting via ₃-adrenoceptors) was an agonist with an intrinsic activity greater than CL316243 [GenBank] . In mouse ileum, SR59230A relaxed smooth muscle, although concentration-response curves were biphasic. Relaxant effects were produced by concentrations that did not affect cAMP levels. Differences in tissue responses to SR59230A were not caused by major differences in expression of Gs. ECAR responses were not affected by pretreatment of cells with pertussis toxin, indicating that signaling did not involve Gi. Therefore, SR59230A displays agonist and antagonist actions at the mouse ₃-adrenoceptor. Because SR59230A only antagonized accumulation of cAMP in 3T3-F442A adipocytes yet in the same cells was an agonist for ECAR, cAMP-independent signaling pathways must mediate part of the agonist actions in the microphysiometer.

Unlike ₁- and ₂-adrenoceptors, which are intronless, all ₃-adrenoceptors thus far described contain introns (Granneman et al., 1992). In the case of the mouse ₃-adrenoceptor, alternative splicing results in the expression of two splice variants, the _3a- and _3b-adrenoceptor (Evans et al., 1999). Functional studies of the _3a- and _3b-adrenoceptor expressed in Chinese hamster ovary (CHO-K1) cells, using either extracellular acidification rate (ECAR) in the cytosensor microphysiometer or cAMP generation as bioassays, have shown that the _3a- and _3b-adrenoceptor couple to Gs, whereas the _3b-adrenoceptor also couples to Gi (Hutchinson et al., 2002). Both splice variants also couple to extracellular signal-related kinases 1 and 2 (ERK1/2) by a mechanism that does not involve either the generation of cAMP or activation of Gi, suggesting that additional pathways can be activated that are not linked sequentially to the classic pathways (Hutchinson et al., 2002). Therefore, it is possible that the study of a variety of ₃-adrenoceptor ligands may reveal differences in intrinsic activity (IA) at the different signaling pathways and thus shed light on the mechanisms involved.

SR59230A was the first selective ₃-adrenoceptor antagonist described (Manara et al., 1996) and has been shown to competitively antagonize ₃-adrenoceptor-mediated responses in a wide variety of tissues, with greater preference for ₃-adrenoceptors compared with ₁-/₂-adrenoceptors (Manara et al., 1996; Nisoli et al., 1996). However, recent reports indicate that SR59230A also interacts with other -adrenoceptors (Candelore et al., 1999; Brahmadevara et al., 2001; Hutchinson et al., 2001; Yamanishi et al., 2002), and agonist actions have been reported at the cloned human ₃-adrenoceptor (Strosberg and Pietri-Rouxel, 1997; Candelore et al., 1999) and ₃-adrenoceptor in some rodent tissues (Dumas et al., 1998; Brahmadevara et al., 2001; Horinouchi and Koike, 2001).

In this study, we examined the pharmacologic properties of SR59230A at the cloned mouse ₃-adrenoceptor expressed in CHO-K1 cells and the endogenous ₃-adrenoceptor expressed in mouse 3T3-F442A adipocytes or ileum. With respect to its effects on cAMP accumulation, SR59230A behaved as a classic partial agonist relative to CL316243 [GenBank] , with agonist properties being more pronounced as levels of receptor expression increased. However, at low levels of expression in CHO-K1 cells and mouse 3T3-F442A adipocytes that endogenously express ₃-adrenoceptor, SR59230A behaved as a full agonist for ECAR, in some cases exceeding the maximal response attained by CL316243 [GenBank] while acting as a competitive antagonist in the same preparations for cAMP accumulation. The ECAR responses in CHO-K1 cells at all levels of expression and in 3T3-F442A adipocytes were not affected by pretreatment of cells with pertussis toxin. These results suggest that the agonist actions of SR59230A in the cytosensor microphysiometer are not mediated by increases in cAMP levels or activation of Gi-coupled mechanisms.

	Materials and Methods

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Expression of the Mouse ₃-Adrenoceptors in CHO-K1 Cells. The mouse ₃-adrenoceptor (_3a-adrenoceptor) (Evans et al., 1999) was cloned and stably transfected into CHO-K1 cells as previously described (Hutchinson et al., 2002).

Cell Culture. CHO-K1 cells were grown in 50:50 Dulbecco's modified Eagle's medium (DMEM): Ham's F-12 medium containing 10% (v/v) heat-inactivated fetal bovine serum (FBS) as previously described (Hutchinson et al., 2002). 3T3-F442A preadipocytes were maintained in DMEM supplemented with 10% (v/v) iron-fortified calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 U/ml fungizone under 5% CO₂ at 37°C. Preadipocytes were not allowed to reach >60% confluence under subculturing conditions. Upon reaching 90% confluence, 3T3-F442A cells were differentiated into adipocytes by culturing in the aforementioned medium (substituting the iron-fortified calf serum with FBS) containing 5 µg/ml insulin for 7 days, with media changes every 2 days. By day 7, >90% of the cells had differentiated into adipocytes, identified by accumulation of cytoplasmic lipid.

Mouse ileal smooth muscle strips were prepared using the following protocol. Mice (FVB male, 8–14 weeks old) were anesthetized with 80% CO₂/20% O₂ and decapitated. A 10- to 15-cm segment of the small intestine finishing 2 to 3 cm above the ileocecal junction was removed, and its contents were flushed out with warm (37°C) Krebs-Henseleit buffer [118.4 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄.7H₂O, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 11 mM glucose, and 2.5 mM CaCl₂ (pH 7.4)] containing 0.1 mM ascorbic acid and 0.04 mM EDTA and then with Hanks' buffer containing 200 mM L-glutamine. Tissues were pinned out on a Petri dish containing Hanks' balanced salt solution, and the mesenteric fat was carefully dissected away. The tissues were then cut open along the mesenteric fat line and pinned out as a flat tissue. The mucosa was gently scraped off with a sterile microscope slide. Tissues were then cut into segments of approximately 1 cm in length, washed in DMEM and cultured at 37°C in 8% CO₂ in 12-well plates containing DMEM (with 200 mM L-glutamine) for 2 h.

Receptor Binding Assay. Membranes from mouse ileum or 3T3-F442A adipocytes were prepared, and saturation experiments were performed as previously described (Hutchinson et al., 2000, 2002). Competition binding experiments were performed at room temperature in a total volume of 100 µl in 96-well plates. Homogenate (10–40 µg) was incubated with 500 pM [¹²⁵I]cyanopindolol and a range of concentrations of SR59230A for 60 min in the absence or presence of 1 mM (–)-alprenolol to define nonspecific binding. Experiments were performed in duplicate. All reactions were terminated by rapid filtration using a Packard cell harvester (PerkinElmer Life and Analytical Sciences, Boston, MA) through GF/C filters presoaked for 30 min in 0.5% polyethylenimine. Filters were washed four times with wash buffer [50 mM Tris (pH 7.4); 4°C] and dried, 30 µl of Microscint-O was added, and radioactivity was measured using a Packard Top Count.

Cytosensor Microphysiometer Studies. The cytosensor microphysiometer (Molecular Devices, Sunnyvale, CA) was used to measure ₃-adrenoceptor-mediated increases in ECARs as previously described (Hutchinson et al., 2002). CHO-K1 cells expressing the ₃-adrenoceptor were seeded into 12-mm transwell inserts (Costar, Cambridge, MA) at 5 x 10⁵ cells/cup and left to adhere overnight. In experiments using 3T3-F442A adipocytes, cells (1 x 10⁵) were seeded into 12-mm transwell inserts (treated with poly-D-lysine) for 1 to 2 days before medium was changed to that containing insulin and left to differentiate in the inserts for 7 days before use in the cytosensor. When required, cells were incubated with 100 ng/ml pertussis toxin overnight before use. On the day of the experiment, cells were equilibrated for 2 h (1 h for 3T3-F442A adipocyte cells), and cumulative concentration-response curves to CL316243 [GenBank] or SR59230A were constructed in paired sister cells with each concentration of drug exposed to cells for 14 min (in some experiments, curves were also constructed with bupranolol or L748337). In other experiments with 3T3-F442A cells, concentration-response curves to either CL316243 [GenBank] or SR59230A were conducted in the presence or absence of (–)-propranolol. All drugs were diluted in modified RPMI 1640 medium. All results are expressed as a percentage of the maximal response to CL316243 [GenBank] or SR59230A in a given experiment.

cAMP Accumulation Studies. CHO-K1 cells (1 x 10⁵ cells/well) were grown in 12-well plates in DMEM/Ham's F-12 medium containing 0.5% (v/v) FBS for 2 days. 3T3-F442A adipocytes (1 x 10⁵ cells/well) were differentiated as described above. On the day of the experiment, medium was replaced with one containing 1 mM 3-isobutyl-1-methylxanthine and 1 mM ascorbic acid for 2 h before exposure to CL316243 [GenBank] or SR59230A for 30 min. To examine the antagonist effect of SR59230A on CL316243 [GenBank] -mediated responses, SR59230A was incubated with cells for 10 min before the addition of a single submaximal concentration of CL316243 [GenBank] for 20 min (in 3T3-F442A cells, (–)-propranolol was investigated for antagonist actions on CL316243 [GenBank] using the same protocol). cAMP was extracted as described previously (Hutchinson et al., 2002) and measured using a commercial kit (TRK 432; Amersham Biosciences Inc., Piscataway, NJ). All experiments were performed in duplicate, with n referring to the number of independent experiments.

Segments of mouse ileum were prepared as described previously. After an initial equilibration period of 2 h, medium was replaced with one containing 1 mM 3-isobutyl-1-methylxanthine and 1 mM ascorbic acid for 1 h. Tissues were exposed to drugs for 30 min. After treatment of ileum segments, tissues were blotted dry and snapfrozen in liquid nitrogen and stored at –70°C. cAMP was extracted by homogenization of tissues in 0.8 ml of ice-cold 75% (v/v) ethanol containing 1 mM EDTA. Samples were centrifuged at 2000g for 2 min at 4°C, the supernatant was transferred to a new tube, and cAMP was measured as described previously. Data were normalized for protein (Lowry et al., 1951).

Detection of Gs by Immunoblotting. Total cell lysates from mouse ileum were obtained by homogenization of frozen tissue samples in radioimmunoprecipitation assay buffer [1x phosphate-buffered saline, 1% (v/v) Igepal CA-630, 0.5% (v/v) sodium deoxycholate, 0.1% (v/v) sodium lauryl sulfate, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 1 µg/ml aprotinin] with a Dounce homogenizer. Monolayer cells were lysed directly with radioimmunoprecipitation assay buffer. Cell debris was removed by centrifugation at 10,000g for 10 min at 4°C, and protein concentrations were determined (Lowry et al., 1951). Total cell lysates (40 µg of protein) were electrophoresed on 12% polyacrylamide gels and electrotransferred to Hybond-P membranes (pore size, 0.45 µm; Amersham Biosciences Inc.). After blocking with 5% nonfat dry milk in Tris-buffered saline for 1 h at room temperature, membranes were incubated with anti-Gs antibody (1:1000 dilution; Upstate Biotechnology, Lake Placid, NY) overnight at 4°C and detected using a secondary antibody (horseradish peroxidase-linked anti-rabbit IgG) diluted 1:2000 and enhanced chemiluminescence (Amersham Biosciences Inc.).

Organ Bath Studies. Organ bath studies were performed as described previously (Hutchinson et al., 2001). Tissues were mounted on hooks suspended in jacketed organ baths in 6 ml of Krebs-Henseleit solution containing 0.1 mM ascorbic acid and 0.04 mM EDTA and equilibrated for 30 min. Tissues were contracted with 1 µM carbachol (10–15 min) before cumulative concentration-response curves to either CL316243 [GenBank] or SR59230A were constructed at increments of 0.5 log units until a stable state was observed. At the end of each concentration-response curve, tissues were maximally relaxed with 10 µM papaverine, and all responses were expressed as a percentage of this response.

Data Analysis. [¹²⁵I]cyanopindolol saturation binding isotherms were analyzed via nonlinear regression using GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, CA) using a one-site mass action model to derive estimates of the radioligand dissociation constant (K_D) and maximal density of receptor binding sites (B_max). Competition binding isotherms were analyzed according to a one-site mass action model via nonlinear regression using Prism to derive estimates of competitor potency estimates (IC₅₀ values), which were then converted to K_I values (competitor-receptor dissociation equilibrium constant) (Cheng and Prusoff, 1973). For the functional assays of receptor-mediated cAMP accumulation or changes in ECAR, empirical estimates of agonist potency and intrinsic activity were derived from fitting the data to the Hill equation using Prism:

(1)

where E is the effect, [A] is the concentration of agonist, basal is the minimal asymptotic effect in the absence of agonist, range is the maximal response range (i.e., the difference between the minimum response in the absence of ligand and the maximum response in the presence of ligand), n_H is the Hill slope, and EC₅₀ is the midpoint location (potency) parameter. Additional analyses of cAMP experiments performed in CHO cells were undertaken to determine functional estimates of the affinity of SR59230A as an agonist. The "comparative method" (Barlow et al., 1967) was used, whereby the SR59230A concentration-response curve was compared with that of a reference full-agonist (CL316243 [GenBank] ) curve constructed in the same cell line. For the analysis, CL316243 [GenBank] data were fitted to eq. 1, whereas the SR59230A concentration-response data were fitted to the following form of the operational model of agonism (Black and Leff, 1983):

(2)

where E_m is the maximum possible response of the cell above basal, K_A is the agonist receptor equilibrium dissociation constant, n is the slope of the transducer function linking occupancy to response, and is the operational definition of intrinsic activity. For this latter analysis, the parameters E_m and n in eq. 2 were shared with the values of range and n_H, respectively, from eq. 1, thus allowing the estimation of K_A and for SR59230A (Leff et al., 1993). The parameter range and n_H are good estimates of E_m and n, respectively, for a full agonist in a given receptor-transducer system. Thus, the full agonist CL316243 [GenBank] provided the reference parameters E_m and n for the determination of the SR59230A K_A and values. This analysis requires global curve fitting with parameters shared between multiple data sets and was performed using a prerelease version of GraphPad Prism 4.0 (GraphPad Software Inc.).

In functional experiments in which SR59230A was an antagonist of CL316243 [GenBank] -mediated responses, K_B values were calculated according to the method of Furchgott (1972). Because these experiments tested the effects of one antagonist concentration, the derived potency estimate is referred to as an apparent K_B value.

In practice, all of the affinity and potency parameters were estimated as negative logarithms. All results are expressed as mean ± S.E.M. Statistical significance was determined using two-way analysis of variance or t test. P values 0.05 were considered significant.

Drugs and Reagents. CL316243 [GenBank] and SR59230A were gifts from Dr. T. Nash (Wyeth-Ayerst, Princeton, NJ) and Dr. L. Manara (Sanofi-Midy, Milan, Italy), respectively. L748337 was a gift from Dr. M. Candelore (Merck, Darmstadt, Germany), and bupranolol was obtained from Schwarz Pharma (Berlin, Germany). Drugs and reagents were purchased as follows: EDTA (Ajax Chemicals, Melbourne, Australia); G418 (geneticin) (Calbiochem, San Diego, CA); L-(+)-ascorbic acid (Merck); 2200 Ci/mmol [¹²⁵I]cyanopindolol (PerkinElmer Life and Analytical Sciences, Boston, MA); and carbachol (carbamylcholine chloride), isobutylmethylxanthine, insulin (from bovine pancreas), papaverine hydrochloride, (–)-propranolol, and poly-D-lysine (Sigma-Aldrich, St. Louis, MO). Cell culture medium and supplements were obtained from Trace Biosciences (Castle Hill, Australia) except for iron-fortified calf serum (CSL, Ltd., Parkville, Australia). All other drugs were of analytical grade.

Stock solutions of SR59230A were prepared in 50% distilled water, 25% ethanol, and 25% dimethyl sulfoxide. Isobutylmethylxanthine was dissolved in dimethyl sulfoxide. All remaining drugs were prepared in distilled water.

	Results

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[¹²⁵I]Cyanopindolol Binding. We have previously reported the generation of CHO-K1 cells expressing the _3a-adrenoceptor at three different levels of receptor density (Hutchinson et al., 2002). [¹²⁵I]cyanopindolol binding occurred in a saturable manner in membranes from cells expressing receptors at high, medium, or low levels (Hutchinson et al., 2002), and the radioligand binding parameters for each of these expression levels are shown in Table 1. Additional [¹²⁵I]cyanopindolol saturation binding assays were undertaken with membranes from cells that endogenously express the mouse ₃-adrenoceptor. As shown in Table 1, the radioligand had comparable affinities for the native ₃-adrenoceptors in the FVB ileum, differentiated 3T3-F442A adipocytes, and cloned _3a-adrenoceptor in the CHO-K1 cells. However, the maximal density of binding sites of the native receptors most closely approximated that in the low-expressing CHO cells (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 [125I]Cyanopindolol saturation binding parameters for cloned and native mouse 3-adrenoreceptors expressed in membranes derived from different cellular backgrounds Parameters derived from nonlinear regression analysis represent the mean ± S.E.M. (n = 3).

Subsequent binding experiments were performed using SR59230A in competition with [¹²⁵I]cyanopindolol. These data are summarized in Table 2, where it can be seen that SR59230A had higher pK_I values in CHO cells expressing recombinant ₃-adrenoceptors compared with the cells and tissues that endogenously express ₃-adrenoceptors. However, when conducted in the presence of 10 µM guanosine 5'-O-(3-thiotriphosphate) (GTPS), the pK_I values were reduced to values that were similar to the pK_A values obtained in the cAMP functional experiments (Table 2), suggesting that the higher pK_I values were caused by G protein trapping.

View this table: [in this window] [in a new window]   TABLE 2 SR59230A affinity estimates derived from radioligand binding and/or functional assays in cells expressing the cloned or native mouse 3-adrenoceptors Parameters derived from nonlinear regression analysis represent the mean ± S.E.M. from the number of experiments indicated in parentheses.

View larger version (15K): [in this window] [in a new window]   Fig. 1. The effect of the 3-adrenoceptor antagonist SR59230A on cAMP accumulation in cells expressing the 3-adrenoceptor at (a) high, (b) medium, or (c) low receptor levels. The results are expressed as the amount of cAMP produced (picomoles) per well. Basal and CL316243 [GenBank] -stimulated cAMP accumulation is also indicated. Each point shows mean ± S.E.M. (n = 3–5).

View larger version (20K): [in this window] [in a new window]   Fig. 3. Interactions between CL316243 [GenBank] and SR59230A in cells expressing the cloned mouse 3-adrenoceptor at (a) medium or (b) low receptor levels to increase extracellular acidification rates in the cytosensor microphysiometer. The results are expressed as a percentage of the maximal response produced to the highest concentration of CL316243 [GenBank] in each experiment. Each point shows mean ± S.E.M. (n = 4–6).

View larger version (22K): [in this window] [in a new window]   Fig. 6. a, SR59230A and CL316243 [GenBank] increase extracellular acidification rates in 3T3-F442A cells. The results are expressed as a percentage of the maximal response produced to the highest concentration of CL316243 [GenBank] in each experiment. Each point shows mean ± S.E.M. (n = 8). b, 300 nM SR59230A competitively antagonizes CL316243 [GenBank] -mediated increases in extracellular acidification in 3T3-F442A adipocytes. (–)-Propranolol (300 nM) failed to antagonize (c) CL316243 [GenBank] - or (d) SR59230A-mediated increases in extracellular acidification rates in 3T3-F442A adipocytes. The results are expressed as a percentage of the maximal response produced to the highest concentration of CL316243 [GenBank] or SR59230A in each experiment. Each point shows mean ± S.E.M. (n = 4).

View this table: [in this window] [in a new window]   TABLE 3 Agonist potency (pEC50) and IA values for effects on cAMP accumulation in CHO-K1 cells expressing the mouse 3a-adrenoceptor at different receptor levels Values are mean ± S.E.M. of the indicated number of experiments. IA is defined as a fraction of the absolute maximal response to CL316243 [GenBank] in each individual experiment.

In experiments that monitored changes in ECAR in CHO-K1 cells, SR59230A also displayed agonist behavior, causing robust concentration-dependent increases in ECAR in all three ₃-adrenoceptor clones examined. However, in stark contrast to its partial agonist effects in the cAMP assays, SR59230A had an intrinsic activity equal to (CHO medium and CHO low) or significantly (P < 0.05) greater (CHO high) than that for CL316243 [GenBank] , determined using sister cells studied in parallel (Fig. 2; Table 4). As expression levels were lowered, the concentration-response curve for each agonist was shifted to the right (Fig. 2; Table 4), and absolute maximal responses to SR59230A and CL316243 [GenBank] also decreased (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 2. Comparison of the agonist effects of SR59230A in the cytosensor microphysiometer to increase extracellular acidification rates performed in parallel with CL316243 [GenBank] -mediated increases in extracellular acidification rates in cells expressing the 3-adrenoceptor at (a) high, (b) medium, or (c) low receptor levels. The results are expressed as a percentage of the maximal response produced to the highest concentration of CL316243 [GenBank] in each experiment. Each point shows mean ± S.E.M. (n = 4–8).

View this table: [in this window] [in a new window]   TABLE 4 Agonist potency (pEC50) and IA values for effects on ECAR at the cloned or native mouse 3-adrenoceptor Values are mean ± S.E.M. of the indicated number of experiments. IA is defined as a fraction of the absolute maximal response to CL316243 [GenBank] in each individual experiment.

To examine the antagonist effect of SR59230A on CL316243 [GenBank] -mediated increases in ECAR, CHO-K1 cells expressing low or medium levels of receptor were exposed to 300 nM SR59230A for 1 h before construction of CL316243 [GenBank] concentration-response curves (Fig. 3). This concentration of SR59230A was chosen because, based on the binding results, it was appropriate for antagonism of ₃-adrenoceptor-mediated responses while producing a submaximal agonist response relative to CL316243 [GenBank] (Fig. 3). In medium- and low-expressing cells, SR59230A caused the expected initial increase in ECAR, but subsequent CL316243 [GenBank] concentration-response curves were shifted to the right in a parallel manner, with no change in the maximal response compared with CL316243 [GenBank] concentration-response curves constructed in the absence of SR59230A. This shift was used to determined apparent pK_B values for SR59230A in both cell lines (Table 2). Similar experiments could not be performed in CHO-K1 cells expressing the highest levels of _3a-adrenoceptors, because low concentrations of SR59230A that would not be expected to appreciably antagonize CL316243 [GenBank] produced significant increases in ECAR in their own right (data not shown). In untransfected CHO-K1 cells, no effects were observed for either CL316243 [GenBank] or SR59230A (up to 10 µM) on cAMP accumulation or ECAR (data not shown).

Investigation of Agonist Effects on Extracellular Acidification Rates by Other ₃-Adrenoceptor Antagonists. To determine whether the agonist properties of SR59230A were unique to this compound or whether other antagonists acting at the ₃-adrenoceptor were also capable of having agonist effects on ECAR, concentration-response curves were constructed with a "human active" ₃-adrenoceptor antagonist, L748337, and a nonspecific -adrenoceptor antagonist, bupranolol, in cells expressing high levels of receptor (Fig. 4; Table 5). L748337 was a full agonist with respect to CL316243 [GenBank] - or SR59230A-mediated responses, whereas bupranolol had no agonist effect with respect to ECAR (preliminary experiments also indicated that propranolol and carvedilol had agonist activity on ECAR). Bupranolol acted as an antagonist, capable of antagonizing CL316243 [GenBank] -, SR59230A-, or L748337-mediated increases in ECAR.

View larger version (25K): [in this window] [in a new window]   Fig. 4. a, CL316243 [GenBank] , the 3-adrenoceptor antagonists SR59230A and L748337, but not the -adrenoceptor antagonist bupranolol increase extracellular acidification rates in cells expressing the cloned mouse 3-adrenoceptor at high receptor levels. The results are expressed as a percentage of the maximal response produced to the highest concentration of CL316243 [GenBank] in each experiment. Each point shows mean ± S.E.M. (n = 4). Bupranolol (100 nM) competitively antagonizes (b) CL316243 [GenBank] -, (c) SR59230A-, or (d) L748337-mediated increases in extracellular acidification in cells expressing the cloned mouse 3-adrenoceptor at high receptor levels. The results are expressed as a percentage of the maximal response produced to the highest concentration of ligand in each experiment. Each point shows mean ± S.E.M. (n = 4).

View this table: [in this window] [in a new window]   TABLE 5 Agonist potency values (pEC50) for two 3-adrenoceptor antagonists and pKB values of bupranolol-mediated antagonism of agonist effects on ECAR at the cloned mouse 3-adrenoceptor at high levels Values are mean ± S.E.M. of n = 4.

Agonist and Antagonist Effects of SR59230A at the Endogenous Mouse ₃-Adrenoceptor Expressed in 3T3-F442A Adipocytes. To determine whether differences in signaling efficiency of SR59230A for the cAMP pathway and ECAR were confined to cloned _3a-adrenoceptors expressed in a recombinant system, subsequent experiments investigated the same bioassays with cells that natively express the mouse ₃-adrenoceptor. When cAMP accumulation was investigated in differentiated 3T3-F442A adipocytes, SR59230A produced no response in contrast to the marked increase of cAMP levels after CL316243 [GenBank] treatment (pEC₅₀, 7.91 ± 0.16; n = 4; Fig. 5). However, SR59230A did bind to the ₃-adrenoceptor in adipocytes because it was able to concentration-dependently inhibit cAMP production in response to a submaximal concentration of CL316243 [GenBank] (30 nM), with a pIC₅₀ value of 7.69 ± 0.41 (n = 3; Fig. 5). Although differentiated 3T3-F442A adipocytes express all three -adrenoceptor subtypes (El Hadri et al., 1996, 1997), CL316243 [GenBank] -mediated increases in cAMP levels were not inhibited by the ₁-/₂-adrenoceptor antagonist (–)-propranolol (300 nM), the ₁-adrenoceptor antagonist CGP20712A (300 nM), or the ₂-adrenoceptor antagonist ICI118551 (300 nM) (data not shown), confirming that CL316243 [GenBank] -mediated increases in cAMP are caused by actions at the ₃-adrenoceptor.

View larger version (19K): [in this window] [in a new window]   Fig. 5. a, SR59230A has no effect on cAMP levels in differentiated 3T3-F442A adipocytes compared with the increase mediated by CL316243 [GenBank] . b, in cells pretreated with SR59230A before stimulation with 30 nM CL316243 [GenBank] , however, SR59230A antagonized CL316243 [GenBank] -mediated increases on cAMP levels. The results are expressed as the amount of cAMP produced (pmol) per well. Each point shows mean ± S.E.M. (n = 3–4).

When ₃-adrenoceptor-mediated changes in ECAR were monitored, a different profile of activity was observed. SR59230A caused concentration-dependent increases in ECAR in differentiated 3T3-F442A adipocytes with an intrinsic activity significantly (P < 0.05) greater than that of CL316243 [GenBank] , determined in sister cells studied in parallel (Fig. 6; Table 4). To further examine the effect of SR59230A on CL316243 [GenBank] -mediated increases in ECAR, 3T3-F442A cells were exposed to 300 nM SR59230A for 1 h before construction of CL316243 [GenBank] concentration-response curves. SR59230A caused an initial increase in ECAR, and subsequent construction of the CL316243 [GenBank] concentration-response curve showed that SR59230A shifted the CL316243 [GenBank] concentration-response curve to the right, with an apparent pK_B value close to that obtained in similar experiments conducted in the transfected CHO cells (Table 2). Maximum responses to CL316243 [GenBank] were not changed by 300 nM SR59230A. To determine whether the agonist actions of SR59230A were caused by actions at ₁-/₂-adrenoceptors that are also expressed in 3T3-F442A adipocytes, concentration-response curves to either CL316243 [GenBank] or SR59230A were conducted in the presence or absence of 300 nM (–)-propranolol. The addition of (–)-propranolol did not alter basal ECAR, and subsequent CL316243 [GenBank] or SR59230A concentration-response curves were not affected by (–)-propranolol (Fig. 6).

Agonist Effects of SR59230A on Extracellular Acidification Rate Are Independent of Gi. Agonist activation of the ₃-adrenoceptor can produce signaling through Gi (Chaudhry et al., 1994; Gerhardt et al., 1999; Soeder et al., 1999; Hutchinson et al., 2002), and although there is evidence that the _3a-adrenoceptor variant used in the present studies couples solely to Gs (Hutchinson et al., 2002), it is feasible that SR59230A may activate the Gi pathway. Pertussis toxin pretreatment of CHO-K1 cells expressing high, medium, or low levels of _3a-adrenoceptor had no effect on the ECAR response to SR59230A (Fig. 7). Although 3T3-F442A adipocytes express _3a-adrenoceptors and the pertussis toxin-sensitive _3b-adrenoceptor (D. S. Hutchinson, unpublished observations), pertussis toxin pretreatment had no significant effect on responses to SR59230A, suggesting that the ECAR response to SR59230A is not mediated by Gi.

View larger version (24K): [in this window] [in a new window]   Fig. 7. The effect of pertussis toxin pretreatment (100 ng/ml, 16 h) on extracellular acidification rate responses in CHO-K1 cells expressing (a) high, (b) medium, and (c) low levels of mouse 3a-adrenoceptors and in (d) 3T3-F442A cells that endogenously express mouse 3-adrenoceptors. The results are expressed as a percentage of the maximal response produced to the highest concentration of SR59230A in control-treated cells in each experiment. Each point shows mean ± S.E.M. (n = 3–4).

SR59230A-Mediated Relaxation of Mouse Ileum Is Independent of Increases in cAMP Levels. The findings in the cell lines suggested that SR59230A could produce agonist responses via the ₃-adrenoceptor in recombinant and natively expressing cells by a mechanism unrelated to its ability to signal via the cAMP pathway. This hypothesis was further investigated using mouse ileum. SR59230A produced a concentration-dependent relaxation of mouse ileum that was biphasic (Fig. 8). The first phase (pEC₅₀, 7.97 ± 0.30; n = 6) occurred at concentrations similar to those reported previously for (–)-isoprenaline (Hutchinson et al., 2001) and CL316243 [GenBank] (pEC₅₀, 7.57 ± 0.53; n = 5) and caused a response that was approximately 40% of the maximum relaxation to papaverine. The second phase failed to reach a maximum within the concentration range used. Investigation of cAMP accumulation in this tissue provided no evidence for an involvement of this second messenger in the actions of SR59230A. cAMP accumulation in ileum (n = 6 or 7) was significantly increased compared with basal levels (68.07 ± 15.45 pmol of cAMP/mg protein) by the direct activator of adenylate cyclase, forskolin (100 µM; 1392.40 ± 195.98 pmol of cAMP/mg protein; P < 0.0001); the -adrenoceptor agonist (–)-isoprenaline (10 µM; 177.82 ± 36.79 pmol of cAMP/mg protein; P < 0.01); the ₃-adrenoceptor agonist CL316243 [GenBank] (10 µM; 144.49 ± 23.11 pmol of cAMP/mg protein; P < 0.01); but not by SR59230A (10 µM; 75.90 ± 18.43 pmol of cAMP/mg protein; N.S.).

View larger version (17K): [in this window] [in a new window]   Fig. 8. SR59230A and CL316243 [GenBank] relax mouse ileum precontracted with carbachol in a concentration-dependent manner. Each point shows mean ± S.E.M. (n = 5–6).

Comparison of Gs Levels in Cells and Tissues by Immunoblotting. Because differences in signaling observed between CHO cells, 3T3-F442A adipocytes, and ileal smooth muscle could be caused by differences in the levels of Gs proteins, Western blot analyses were carried out using an antibody that recognizes the long and short isoforms of Gs. In all of the tissues, the dominant isoform present was the short Gs isoform, and it was expressed at similar levels in CHO cells, 3T3-F442A adipocytes, and ileal smooth muscle (Fig. 9), suggesting that the different signaling properties observed do not result from major differences in Gs levels.

View larger version (18K): [in this window] [in a new window]   Fig. 9. Immunoblot of Gs small and large isoforms in CHO-K1 cells expressing high, medium, and low levels of 3a-adrenoceptors, 3T3-F442A adipocytes, and mouse ileum. The dominant isoform present was the small isoform, which was expressed at similar levels in all of the tissues.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
This study reveals that the ₃-adrenoceptor antagonist SR59230A possesses agonist properties at cloned and natively expressed mouse ₃-adrenoceptors. The striking disparity between the effects of SR59230A and CL316243 [GenBank] when ECAR is used as the bioassay rather than cAMP accumulation provides strong evidence for pleiotropic coupling of the ₃-adrenoceptor to distinct signaling pathways that are differentially activated by the two ligands.

In initial experiments, SR59230A competed with [¹²⁵I]cyanopindolol binding (Table 2) with affinity appropriate for binding to ₃-adrenoceptors (Candelore et al., 1999; Hutchinson et al., 2002). However, pK_I values in cells expressing recombinant ₃-adrenoceptors were greater than those determined in 3T3-F442A cells and mouse ileum. Because the binding assays were conducted in membrane homogenates, it is likely that receptor-G protein complexes would be present (Christopoulos and El-Fakahany, 1999), and assuming that total cellular G protein content was not limiting, this would cause an overestimation of apparent agonist affinity that would be greatest in cells expressing high receptor levels (Christopoulos and El-Fakahany, 1999). The reduction in pK_I values after the addition of guanosine 5'-O-(3-thiotriphosphate) supported this view.

The affinity of SR59230A also was determined using the concentration-response curve to the full agonist CL316243 [GenBank] in cAMP assays, and operational model fitting of SR59230A concentration-response curves allowed functional estimates of agonist affinity to be obtained (Table 2). The pK_A estimates agreed with pK_I estimates determined from binding assays, suggesting that the impact of G protein trapping in the membrane-based binding assays was minimal and restricted to the high-expressing cells.

Interestingly, the use of SR59230A as an antagonist of CL316243 [GenBank] -mediated increases in ECAR to estimate affinity yielded the largest discrepancies compared with the other two methods. The pK_B values for SR59230A were some 10-fold higher than pK_I or pK_A values from binding or cAMP assays (Table 2). It is possible that agonist properties of SR59230A during the initial 1-h equilibration led to a partial desensitization of CHO-K1-₃ cells to CL316243 [GenBank] . This would underestimate CL316243 [GenBank] potency in the presence of SR59230A and hence overestimate apparent affinity. Further experiments are required to examine this possibility.

SR59230A increased cAMP in CHO-K1-₃ cells and was a partial agonist relative to CL316243 [GenBank] (Fig. 1; Table 3), with the effect depending on receptor expression; the reduction in receptor density across the three cell lines was associated with a corresponding reduction in agonist potency and intrinsic activity of SR59230A and CL316243 [GenBank] , as predicted by classic receptor theory (Christopoulos and El-Fakahany, 1999). In contrast, no agonist effects of SR59230A were detected on cAMP accumulation in mouse ileum or 3T3-F442A adipocytes, which endogenously express the mouse ₃-adrenoceptor. Given that our binding experiments show that these cells express ₃-adrenoceptors at similar levels to CHO-K1-₃ low-expressing cells (Table 1), the agonist activity of SR59230A in CHO-K1-₃ cAMP assays and the absence of such activity in ileum or 3T3-F442A cells suggest that stimulus-response coupling in adipocytes and ileum is significantly weaker than in CHO-K1-₃ cells. Accordingly, a partial agonist, such as SR59230A, would lose its agonist properties under conditions of reduced stimulus-response coupling relative to the full agonist CL316243 [GenBank] . The differences were not caused by marked differences in the expression of Gs in the different cells because immunoblots showed similar levels of the predominant Gs small isoform in CHO cells, 3T3-F442A cells, and ileum. Importantly, SR59230A antagonized CL316243 [GenBank] -mediated increases in cAMP accumulation in 3T3-F442A cells (Fig. 5), confirming that SR59230A still bound to ₃-adrenoceptors in these cells.

In contrast to cAMP accumulation, a strikingly different signaling profile was observed for SR59230A but not CL316243 [GenBank] when function was measured using the cytosensor microphysiometer. Although SR59230A had significantly lower potency than CL316243 [GenBank] , it was a full agonist for ECAR, with an intrinsic activity somewhat greater than CL316243 [GenBank] (Fig. 2; Table 4). This was not restricted to CHO-K1-₃ cells because the maximal ECAR response to SR59230A in 3T3-F442A adipocytes was greater than that for CL316243 [GenBank] (Fig. 6). The ECAR response to SR59230A was not caused by low levels of ₁-/₂-adrenoceptors in differentiated 3T3-F442A adipocytes (El Hadri et al., 1997), because the ₁-/₂-adrenoceptor antagonist (–)-propranolol failed to shift the concentration-response curves to either CL316243 [GenBank] or SR59230A in these cells.

These observations led us to consider mechanisms to account for such an effect. A higher intrinsic activity may indicate higher intrinsic activity for SR59230A than CL316243 [GenBank] ; however, cAMP assays showed that SR59230A is a lower intrinsic activity agonist than CL316243 [GenBank] . This property would be retained if the ECAR were linked solely to cAMP (i.e., down-stream signal amplification could lead to enhancement of the intrinsic activity of SR59230A but not to a greater maximal response than CL316243 [GenBank] if both agonists activate the same signal transduction cascade). It is also possible that CL316243 [GenBank] activates Gs and Gi to mediate the responses detected as a change in ECAR, whereas SR59230A only activates Gs. The net result would be a greater maximal response to agonist signaling solely through the stimulatory pathway; however, the mouse _3a-adrenoceptor, as used here, cannot couple to Gi (Hutchinson et al., 2002). Therefore, it is more likely that SR59230A couples to another signaling pathway, independently of its effects on cAMP, that is either not used or only weakly recruited by CL316243 [GenBank] . In contrast to linked intracellular signaling cascades, which predict increases in potency and intrinsic activity of agonists with increases in signal amplification, the summation of multiple independent signaling pathways that converge to a common endpoint, such as ECAR, can account for an enhanced response range to an agonist without postulating the need for increased agonist potency. This also agreed with our experimental observations. If ECAR were linked to upstream changes in cAMP accumulation, the degree of signal amplification required to increase the SR59230A maximal response from that of a partial agonist in the cAMP assay to a full agonist in the cytosensor assay would also cause significant enhancement in agonist potency. However, a comparison of pEC₅₀ values obtained in the CHO-K1 cells (Tables 3 and 4) clearly indicates that this is not the case.

Experiments using mouse ileum also provided evidence for relaxation responses to SR59230A not involving cAMP. SR59230A caused relaxation of the ileum significantly greater than that caused by CL316243 [GenBank] (Fig. 8), but only the latter agent caused increases in cAMP. Horinouchi and Koike (2002) also suggested that ₃-adrenoceptor-mediated relaxation of gastrointestinal tissues occurs independently of cAMP.

Currently, the pathway stimulated by SR59230A to increase ECAR is unknown. The ₃-adrenoceptor also signals through Gi to inhibit cAMP production or activate ERK1/2, c-Src, p38 mitogen-activated protein kinase, and the nitricoxide synthase pathway to increase cGMP production (Chaudhry et al., 1994; Gerhardt et al., 1999; Soeder et al., 1999; Trochu et al., 1999; Cao et al., 2000, 2001; Lindquist et al., 2000; Hutchinson et al., 2002; Mizuno et al., 2002). Because the response was insensitive to pertussis toxin, Gi would not seem to be involved, but it is feasible that SR59230A may activate one or more of the remaining signaling pathways. In guinea pig duodenum and gastric fundus endogenously expressing ₃-adrenoceptors, SR59230A is an agonist with a potency similar to isoprenaline (Horinouchi and Koike, 2001). At high concentrations, it causes relaxation of hypoxic pulmonary vasoconstriction in rats (Dumas et al., 1998) and rat aortic rings (Brahmadevara et al., 2003). We are further investigating the mechanism of SR59230A-mediated increases in ECAR.

Other -adrenoceptor antagonists have agonist actions at ₃-adrenoceptors, including tertatolol, alprenolol, propranolol, oxprenolol, pindolol, cyanopindolol, nadolol, and carteolol (Arch, 2000). We have shown herein that in CHO-K1-₃ cells, the ₃-adrenoceptor antagonist L748337 also has agonist actions on ECAR, whereas bupranolol is a classic antagonist of CL316243 [GenBank] -, SR59230A-, or L748337-mediated responses. Clearly, the agonist effects are not confined to SR59230A, and further analysis of other -adrenoceptor antagonists may provide information on structural requirements for pleiotropic signaling.

Several reports suggest that receptors exist in multiple active states corresponding to different conformations with specific pharmacologic and functional properties and that different ligands and/or G proteins affect these states in different ways (Kenakin, 2002). For the human ₃-adrenoceptor transfected in CHO-K1 cells, it has been suggested that three different states exist: one state favoring ₃-adrenoceptor signaling toward adenylate cyclase, another favoring ERK1/2, and other receptor states not discriminating between the two signaling pathways (Gerhardt et al., 1999). This may also occur in mouse CHO-K1-₃ cells, because pEC₅₀ values for ECAR, cAMP accumulation, and ERK1/2 activation are markedly different when the receptor is stimulated with CL316243 [GenBank] (Hutchinson et al., 2002).

In conclusion, SR59230A displayed agonist properties at the mouse ₃-adrenoceptor that were increased with the level of receptor expression. However, SR59230A displayed higher intrinsic activity in ECAR than in cAMP accumulation bioassays. These differences were further accentuated in 3T3-F442A cells, in which SR59230A acted as a classic competitive antagonist for cAMP responses produced by CL316243 [GenBank] but as a full agonist with an intrinsic activity greater than CL316243 [GenBank] in the cytosensor microphysiometer. Therefore, SR59230A is capable of producing agonist responses in 3T3-F442A cells by a mechanism independent of cAMP.

	Footnotes

 
This work was supported by the National Health and Medical Research Council of Australia. D.S.H. was a Monash University Postgraduate Scholar and was supported by the Monash University Postgraduate Publications Award. M.S. is a Monash University Postgraduate Scholar. A.C. is a Senior Research Fellow of the National Health and Medical Research Council of Australia.

doi:10.1124/jpet.104.076901.

ABBREVIATIONS: CGP12177A, 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one; CL316243 [GenBank] , (R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate; BRL37344 sodium 4-{2-[2-hydroxy-2-(3-chlorophenyl)-ethylamino]propyl}phenoxyacetate sesquihydrate; SR59230A, 3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate; CHO, Chinese hamster ovary; ECAR, extracellular acidification rate; ERK, extracellular signal-related kinase; IA, intrinsic activity; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GTPS, guanosine 5'-O-(3-thiotriphosphate); L748337, (S)-N-[4-[2-[[3-[-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzene sulfonamide; CGP20712A, (±)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate; ICI118551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol.

¹ Current address: The Werner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, Stockholm, Sweden.

Address correspondence to: Dr. Roger J. Summers, Department of Pharmacology, P.O. Box 13E, Monash University, Victoria 3800, Australia. E-mail: roger.summers{at}med.monash.edu.au

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