QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: mol.107.035337v1 72/5/1359    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Related articles in MolPharm Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sato, M. Articles by Summers, R. J. Search for Related Content PubMed PubMed Citation Articles by Sato, M. Articles by Summers, R. J.

Ligand-Directed Signaling at the ₃-Adrenoceptor Produced by 3-(2-Ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate (SR59230A) Relative to Receptor Agonists

Department of Pharmacology, Monash University, Victoria, Australia

Received February 24, 2007; accepted August 22, 2007

	Abstract

Top Abstract References

 
This study examines signaling pathways activated by the mouse ₃-adrenoceptor (AR) expressed in Chinese hamster ovary cells at high (CHO₃H) or low (CHO₃L) levels. Functional responses included extracellular acidification rate (ECAR), cAMP accumulation, and p38 mitogen-activated protein kinase (MAPK) or extracellular signal-regulated protein kinase 1/2 (Erk1/2) phosphorylation. (–)-Isoproterenol and the ₃-AR agonist (R, R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-decarboxylate (CL316243 [GenBank] ) caused concentration-dependent increases in cAMP accumulation and ECAR in CHO₃H and CHO₃L cells. For cAMP accumulation, the ₃-AR ligand SR59230A was a partial agonist in CHO₃H and an antagonist in CHO₃L cells but for ECAR was an agonist at both expression levels. This suggested that SR59230A, which is normally regarded as an antagonist, can selectively activate pathways leading to ECAR. Examination of the pathways stimulated by (–)-isoproterenol, CL316243 [GenBank] , and SR59230A for both ECAR and cAMP accumulation suggested that the cAMP pathway predominates in CHO₃H cells, whereas p38 MAPK is a major contributor to ECAR in CHO₃L cells and was the sole contributor to responses to SR59230A. Western blots of p38 MAPK and Erk1/2 phosphorylation confirmed that MAPKs are activated in CHO₃H and CHO₃L cells by CL316243 [GenBank] and SR59230A but that SR59230A has much higher efficacy. In addition, p38 MAPK phosphorylation displayed differences in drug potency and efficacy between CHO₃H and CHO₃L cells related to inhibition of the response by cAMP. Thus, CL316243 [GenBank] and SR59230A display reversed orders of efficacy for cAMP accumulation compared with Erk1/2 and p38 MAPK phosphorylation, providing a strong indication of ligand-directed signaling.

It is possible that the ability of ligands such as SR59230A to block cAMP accumulation in response to ₃-AR agonists and yet activate other pathways represents an example of ligand-directed signaling. There are now a number of examples of ligand-directed signaling (Urban et al., 2007) that have been explained by the existence of multiple active conformations of receptors, termed the "conformational cafeteria" (Kenakin, 2003; Clarke, 2005). Several recent studies have described the stimulation of Erk1/2 phosphorylation by -AR ligands usually classified as antagonists in cells expressing ₁or ₂-ARs (Azzi et al., 2003; Baker et al., 2003; Galandrin and Bouvier, 2006). The inverse agonists ICI118551 and propranolol are antagonists for cAMP responses but agonists for Erk1/2 activation (Azzi et al., 2003; Baker et al., 2003). A recent study that examined the effects on Erk1/2 phosphorylation of a wide range of -AR ligands after activation of ₁or ₂-ARs found complex efficacy profiles, with compounds that acted as inverse agonists for the cAMP pathway displaying agonist, neutral antagonist, or inverse agonist properties with respect to Erk1/2 activation (Galandrin and Bouvier, 2006). There were also compounds that acted as partial agonists for cAMP accumulation but were agonists or neutral antagonists for Erk1/2 phosphorylation (Galandrin and Bouvier, 2006).

In this study, we have examined signaling pathways used by the ₃-AR in response to stimulation by the -AR agonist (–)-isoproterenol, the selective ₃-AR agonist CL316243 [GenBank] , and the ₃-AR ligand SR59230A in cells expressing high (CHO₃H) and low (CHO₃L) levels of the mouse ₃-AR. We find that (–)-isoproterenol and CL316243 [GenBank] produce equivalent responses for cAMP accumulation in both lowand high-expressing cells, whereas SR59230A is a partial agonist in high-expressing cells and a competitive antagonist of responses to CL316243 [GenBank] in low-expressing cells. All three ligands are agonists for ECAR in both lowand high-expressing cells. The agonist efficacy with respect to CL316243 [GenBank] and SR59230A is reversed for p38 MAPK and Erk1/2 phosphorylation. In both lowand high-expressing cells, SR59230A stimulates Erk1/2 phosphorylation with much higher efficacy than CL316243 [GenBank] . Although similar in pattern to Erk1/2, p38 MAPK phosphorylation shows differences in drug potency and efficacy between CHO₃H and CHO₃L cells related to the inhibition of the response by cAMP. SR59230A therefore preferentially directs signaling to MAPK pathways.

Materials and Methods
Expression of the Mouse ₃-AR in CHO-K1 Cells. Inserts carrying the coding region of the _3a-AR were created as described previously (Hutchinson et al., 2002). Plasmids were linearized with ScaI before transfection. Fifteen micrograms of each plasmid was transfected into 5 x 10⁶ CHO-K1 cells by electroporation (270 V, 960 µF) in a Gene Pulser II (Bio-Rad Laboratories, Hercules, CA). The cells were grown for 48 h, and then stable transformants were selected in medium containing 800 µg/ml G418. Clonal cell lines were obtained by limiting dilution of mixed cell populations, and clones were expanded and analyzed for expression levels by a single point [¹²⁵I](–)-cyanopindolol (800 pM) binding screen. Suitable clones were grown up for a full saturation binding analysis.

Cell Culture and Treatments. CHO-K1 cells were grown as monolayers in 50:50 Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium containing 10% (v/v) fetal bovine serum (FBS), glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml). Clonal CHO-K1 lines transfected with the ₃-AR were grown in the above media but with the addition of G418 (400 µg/ml). Cells were maintained under 5% CO₂ at 37°C, and cells were passaged every 3 to 4 days.

Radioligand Binding Assay. Cell membranes were prepared as described earlier (Hutchinson et al., 2002), and saturation binding experiments were performed (Hutchinson et al., 2002). In brief, homogenate (10–20 µg of protein) was incubated with [¹²⁵I](–)-cyanopindolol (100–2000 pM) for 60 min at room temperature in the absence or presence of (–)-alprenolol (1 mM) to define nonspecific binding. Reactions were terminated by rapid filtration through GF/C filters presoaked for 30 min in 0.5% (v/v) polyethylenimine using a Packard Cell Harvester, and radioactivity was measured using a TopCount liquid scintillation analyzer (PerkinElmer Life and Analytical Sciences, Waltham, MA). Experiments were performed in duplicate; n refers to the number of different membrane homogenate samples used.

cAMP Accumulation Studies. Cells (1 x 10⁴/well) were grown in 96-well plates in DMEM/Ham's F-12 medium containing 0.5% (v/v) FBS for 2 days. On the day of experiment, the medium was aspirated, and appropriate drugs were diluted in stimulation buffer [1 mg/ml bovine serum albumin (BSA), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and 0.5 M HEPES, pH 7.4, in Hanks' balanced salt solution] added in a final volume of 100 µl. After 30 min of incubation at 37°C, the medium was removed, and 100 µl of lysis buffer [1 mg/ml BSA, 0.3% (v/v) Tween 20, 0.5 M HEPES, and 0.5 mM IBMX, pH 7.4] was added. Samples were rapidly frozen at –70°C and then thawed before assay to lyse cells before measurement of cAMP.

In experiments examining the effect of inhibitors, cells were treated with inhibitors for 30 min before stimulation with appropriate drugs. cAMP accumulation was measured using the cAMP Alphascreen method (PerkinElmer, Victoria, Australia). Samples were defrosted, and cAMP standards (10 pM to 1 µM) were prepared in detection buffer [0.4% (v/v) Hanks' balanced salt solution, 3 mM HEPES, 0.2% (v/v) Tween 20, and 0.1% (v/v) BSA, pH 7.4], and 5 µl of unknown samples or cAMP standards were transferred into a white 384-well plate. Five microliters of acceptor beads (anti-cAMP acceptor beads diluted in detection buffer) was aliquoted to each well and incubated for 30 min in the dark. Donor bead mix (15 µlof streptavidin donor beads diluted in detection buffer, 133 units/ml biotinylated cAMP) solution was added to each well, and the plate was sealed and incubated in the dark overnight. cAMP accumulation was detected using a Fusion microplate reader (PerkinElmer). cAMP responses were expressed as a percentage of the response to 100 µM forskolin to correct for variability in cell number or viability between individual samples. The level of receptor expression influenced absolute cAMP accumulation to forskolin [42 ± 5.2 (8) pmol/well CHO₃H and 27 ± 1.5 (11) pmol/well CHO₃L]. This difference does not influence the interpretation of the data, because the comparisons are between the agonists and SR59230A, not between cells with high or low ₃-AR expression. In fact, making the additional correction for absolute forskolin response would accentuate differences between agonist and SR59230A-stimulated cAMP accumulation in highand low-expressing cells.

Cytosensor Microphysiometer Studies. The cytosensor microphysiometer (Molecular Devices, Sunnyvale, CA) was used to measure ₃-AR-mediated increases in ECAR as described previously (Hutchinson et al., 2002, 2005). In brief, CHO-K1 cells expressing the ₃-AR were seeded into 12-mm transwell inserts (Costar; Corning Life Science, Acton, MA) at 5 x 10⁵ cells/cup and left to adhere overnight. On the day of the experiment, cells were equilibrated for 2 h, and cumulative concentration-response curves to (–)-isoproterenol, CL316243 [GenBank] , or SR59230A were constructed in paired sister cells with each concentration of drug exposed to cells for 14 min. Results are expressed as a percentage of the maximal response to (–)-isoproterenol. In experiments examining the effect of inhibitors, cells were treated with inhibitors for 30 min before stimulation with appropriate drugs for 30 min. All drugs were diluted in modified RPMI 1640 medium. These results are expressed as a percentage of the maximal response to (–)-isoproterenol, CL316243 [GenBank] , or SR59230A over basal.

Western Blotting. Cells were grown in 12-well plates at 1 x 10⁵/well in DMEM/Ham's F-12 medium containing 0.5% FBS for 2 days, and the medium was replaced (to 0% FBS) 2 h before the experiment. In time-course studies, cells were exposed to agonist for 0 to 30 min. Cells were lysed directly in each well by the addition of 40 µl of 65°C SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromphenol blue). Cells were scraped, transferred to an Eppendorf tube on ice, and sonicated for 10 s followed by heating to 95°C for 5 min. Aliquots of the samples were separated on a 12% polyacrylamide gel and electrotransferred to a Hybond-C Extra nitrocellulose membrane (pore size, 0.45 mm; GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) with a semidry electroblotter. After transfer, the membranes were allowed to soak in Tris-buffered saline for 5 min, followed by quenching of nonspecific binding (1 h at room temperature in 5% nonfat dry milk, 0.1% Tween 20 in Tris-buffered saline). Membranes were incubated overnight at 4°C with primary antibody, phospho-p38 MAPK (Thr180/Tyr184), or phospho-p44/42 MAPK (Thr202/Tyr204), diluted 1:1000. This was detected using a secondary antibody (horseradish peroxidase-linked anti-rabbit IgG diluted 1:2000) and enhanced chemiluminescence. The membranes were then stripped with 10 M urea, 50 mM sodium phosphate, 100 mM -mercaptoethanol for 30 min at 50°C, and reprobed with the appropriate p38 MAPK or p44/42 MAPK antibody, detected with the same secondary antibody. Results are expressed as the ratio of phosphorylated to total p38 MAPK or Erk1/2 protein. All experiments were performed in duplicate with n referring to the number of independent experiments performed.

Data Analysis. All results were expressed as a mean ± S.E.M. of n experiments. Data were analyzed using nonlinear curve-fitting (Prism version 4.0; GraphPad Software Inc., San Diego, CA). Concentration-response curves were analyzed using the general equation for a sigmoid curve with a Hill slope of 1: Y = Bottom + Top–Bottom/^{1 + 10 log EC50 – X}, where Y is the response, X is the log [ligand], Bottom is the Y response value for the bottom plateau, Top is the Y response value for the top plateau, and EC₅₀ is the ligand concentration corresponding to the Y value halfway between bottom and top. Statistical significance was determined using two-way analysis of variance tests or Student's t test. Probability values less than or equal to 0.05 were considered significant.

Drugs and Reagents. RWJ67657 was kindly supplied by Dr John Siekierka (Johnson & Johnson, Raritan, NJ). Drugs and reagents were purchased as follows: G418, LY294002, PP2, and PD98059 were from CalBiochem Corporation (La Jolla, CA); (–)-[¹²⁵I]CYP (2200 Ci/mmol) was from Perkin Elmer; (–)-alprenolol, bacitracin, IBMX, polyethylenimine, (–)-isoproterenol, CL316243 [GenBank] , SR59230A, forskolin, 2', 3'-dideoxyadenosine (DDA), and H-89 were from Sigma Chemical Co. (St. Louis, MO); aprotinin, leupeptin, and pepstatin A were from Valeant Pharmaceuticals (Costa Mesa, CA). All cell culture media and supplements were obtained from Trace Biosciences (Castle Hill, NSW, Australia). Antibodies were obtained from Cell Signaling Technology (Danvers, MA). All other drugs and reagents were of analytical grade.

Results
Radioligand Binding Studies. Stably transfected CHO-K1 cells were examined for levels of receptor expression in saturation experiments using [¹²⁵I]CYP. The pK_D values and expression levels for high-(CHO₃H) and low (CHO₃L)-expressing cells were 9.0 ± 0.3 and 1150 ± 240 fmol/mg of protein and 9.5 ± 0.1 and 115 ± 6 fmol/mg of protein, respectively (n = 4). Because receptors expressed at high and low levels had similar pK_D values, this suggested that the level of expression had little or no effect on [¹²⁵I]CYP binding affinity. It is also known from previous studies (Hutchinson et al., 2002) that the G protein-coupling properties of ₃-ARs are retained over a wide range of expression levels and are not the result of receptor overexpression.

Effects of (–)-Isoproterenol, CL316243 [GenBank] , and SR59230A on cAMP Accumulation in Cells Expressing ₃ Adrenoceptors at High and Low Levels. In CHO-K1 cells stably expressing high levels of the mouse ₃-AR (CHO₃H), (–)-isoproterenol and the selective ₃-AR agonist CL316243 [GenBank] had similar efficacy in promoting cAMP accumulation. SR59230A behaved as a partial agonist, with a maximum response approximately 70% of that seen with the other agonists (Fig. 1a). CL316243 [GenBank] displayed a 100-fold higher potency than either (–)-isoproterenol or SR59230A (Table 1). Responses in CHO₃L cells were uniformly lower; SR59230A did not promote cAMP accumulation (Fig. 1b) and in fact antagonized CL316243 [GenBank] -mediated increases in cAMP levels in a concentration-dependent manner, with a pK_B value of 7.5 ± 0.3 (n = 4; Fig. 1c). In untransfected CHO-K1 cells, no effects were observed for either CL316243 [GenBank] or SR59230A (up to 10 µM) on cAMP accumulation (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Concentration-response curves for cAMP accumulation in response to (–)-isoproterenol, CL316243, or SR59230A in cells expressing 3-AR at high (a) or low (b) levels. The results are expressed as a percentage of the forskolin (100 µM) response, and each point represents the mean ± S.E.M. (n = 4–8). (–)-Isoproterenol increased cAMP levels (CHO3H max, 28.7 ± 1.4%; CHO3L max, 10.3 ± 0.5%) to a similar extent to CL316243 (CHO3H max, 28.2 ± 1.2%; CHO3L max, 9.3 ± 0.3%). SR59230A behaved as a partial agonist relative to CL316243 in high-expressing cells (max, 20.8 ± 0.7%) but produced no significant cAMP response in CHO3L cells. In c, increases in cAMP accumulation in response to CL316243 (300 nM) were antagonized by SR59230A in CHO3L cells.

View this table: [in this window] [in a new window]   TABLE 1 Comparison of pEC50 values and Vmax values relative to CL316243 for responses in functional bioassays and binding affinity in CHO3H and CHO3L cells Binding affinities are lower than the pEC50 values for the strong agonists isoproterenol and CL316243 and slightly lower for the partial agonist SR59230A. The exceptions were p38 MAPK responses to SR59230A in both low- and high-expressing cells and ECAR in low-expressing cells in which the pEC50 value for the functional responses was lower (markedly so in the high-expressing cells) than the binding affinity (see Discussion).

Effects of (–)-Isoproterenol, CL316243 [GenBank] , and SR59230A on Extracellular Acidification Rate in Cells Expressing ₃ Adrenoceptors at High and Low Levels. Unlike cAMP accumulation, all three drugs produced similar maximal increases in ECAR in both CHO₃H and CHO₃L cells (Fig. 2,a and b). Although the V_max values were similar, SR59230A and (–)-isoproterenol had substantially lower potency than CL316243 [GenBank] , and this difference was equivalent in the highand low-expressing cells (Table 1). In untransfected CHO-K1 cells, no effects were observed for either CL316243 [GenBank] or SR59230A (up to 10 µM) on ECAR (data not shown), confirming that the SR59230A-stimulated ECAR response was ₃-AR-dependent. These data corroborate our previous finding that ECAR responses to SR59230A are not dependent on increases in cAMP levels in low-expressing cells (Hutchinson et al., 2005).

View larger version (21K): [in this window] [in a new window]   Fig. 2. Concentration-response curves for extracellular acidification rate (ECAR) in response to (–)-isoproterenol, CL316243, or SR59230A in cells expressing 3-AR at high (a) or low (b) levels. The results are expressed as a percentage of the maximum response to CL316243. Each point represents the mean ± S.E.M. (n = 4–9). Note that CL316243, (–)-isoproterenol, and SR59230A produce equivalent responses for ECAR at both levels of receptor expression, albeit with differing potency (Table 1).

View larger version (27K): [in this window] [in a new window]   Fig. 3. The effect of inhibitors of the cAMP signaling pathway on ECAR responses to (–)-isoproterenol (), CL316243 (), and SR59230A () in CHO-K1 cells expressing 3-AR at high (a) and low (b) levels. The results are expressed as the percentage of increase from control induced by the agonist over basal ECAR. Each point represents the mean ± S.E.M. (n = 4–7). In CHO3H cells (a), the adenylate cyclase inhibitor DDA (50 µM) substantially inhibited (***, P < 0.001) ECAR responses to 100 nM (–)-isoproterenol, 100 pM CL316243, and 100 nM SR59230A, as did the PKA inhibitor H-89 (10 µM), but in CHO3L cells (b), the same inhibitors had little or no effect on responses to 10 µM(–)-isoproterenol, 50 nM CL316243, and 10 µM SR59230A. Concentrations of (–)-isoproterenol, CL316243, and SR59230A used for the inhibitor studies were determined from concentration-response curves to produce 80 to 90% of maximal responses.

View larger version (38K): [in this window] [in a new window]   Fig. 5. The effect of inhibitors of adenylate cyclase (DDA), PKA (H-89), and p38 MAPK (RWJ67657) on cAMP accumulation responses to (–)-isoproterenol (), CL316243 (), and SR59230A () in CHO-K1 cells expressing 3-AR at high (a) and low (b) levels. The results are expressed as a percentage of the forskolin (100 µM) response and each point represents the mean ± S.E.M. (n = 4–6; ***, P < 0.001; **, P < 0.01; *, P < 0.05). In CHO3H cells (a) DDA (50 µM) significantly inhibited cAMP responses to 100 nM (–)-isoproterenol, 100 pM CL316243, and 100 nM SR59230A whereas H-89 (10 µM) enhanced responses and RWJ67657 (10 µM) had no significant effect. In CHO3L cells (b), DDA (50 µM) significantly decreased cAMP accumulation in response to (–)-isoproterenol and CL316243, whereas H-89 (10 µM) increased cAMP accumulation in response to (–)-isoproterenol and CL316243. RWJ67657 (10 µM) decreased cAMP accumulation in response to (–)-isoproterenol and CL316243. cAMP levels in the presence of SR59230A were unaffected by any of the inhibitors.

View larger version (30K): [in this window] [in a new window]   Fig. 4. The effect of inhibitors of PI3K (LY294002), Src (PP2), MEK (PD98059), and p38 MAPK (RWJ67657) on ECAR responses to (–)-isoproterenol (), CL316243 (), and SR59230A () in CHO-K1 cells expressing 3-AR at high (a) and low (b) levels. The results are expressed as a percentage of increase of control induced by each agonist over basal ECAR. Each point represents the mean ± S.E.M. (n = 4–6; ***, P < 0.001; **, P < 0.01; *, P < 0.05). In CHO3H cells (a), LY294002 (10 µM), PP2 (10 µM), and RWJ67657 (10 µM) all significantly inhibited ECAR responses to 100 nM (–)-isoproterenol, 100 pM CL316243, and 100 nM SR59230A, whereas PD98059 (10 µM) had no significant effect. In CHO3L cells (b), ECAR responses to 10 µM(–)-isoproterenol, 50 nM CL316243, and 10 µM SR59230A were unaffected by LY294002, PP2, or PD98059, whereas RWJ67657 caused significant inhibition, particularly of the response to SR59230A.

Interaction between the cAMP and p38 MAPK Pathways. In cells expressing ₃-AR at high levels, the adenylate cyclase inhibitor DDA significantly decreased cAMP accumulation in response to (–)-isoproterenol, CL316243 [GenBank] , and SR59230A (Fig. 5a). In contrast, treatment with the PKA inhibitor H-89 (10 µM) significantly increased cAMP accumulation in response to each of the three drugs. The p38 kinase inhibitor, RWJ67657 (10 µM) produced no significant effect on cAMP responses at high receptor expression levels. In cells expressing ₃-AR at low levels, DDA significantly decreased cAMP accumulation in response to (–)-isoproterenol and CL316243 [GenBank] , whereas H-89 increased cAMP responses (Fig. 5b). SR59230A did not evoke a cAMP response. It is interesting that RWJ67657 significantly decreased cAMP accumulation in response to (–)-isoproterenol and CL316243 [GenBank] , suggesting that p38 MAPK interacts with adenylate cyclase signaling at this level of expression (Fig. 5b).

To further examine the possible interaction between adenylate cyclase signaling and p38 MAPK, we examined the effect of the cell-permeable cAMP analog 8-bromoadenosine 3', 5'-cAMP (8-Br-cAMP) on p38 MAPK phosphorylation stimulated by sorbitol and SR59230A in CHO₃L cells (Fig. 6). 8-Br-cAMP did not affect basal p38 MAPK phosphorylation. Sorbitol (500 mM) or SR59230A (10 µM) increased p38 MAPK phosphorylation 7or 3-fold, respectively. The sorbitol response was significantly inhibited in the presence of 8-Br-cAMP, and the SR59230A response was completely blocked (Fig. 6).

View larger version (19K): [in this window] [in a new window]   Fig. 6. Interaction between cAMP and p38 MAPK signaling in CHO-K1 cells expressing mouse 3-AR at low levels. p38 MAPK phosphorylation was examined in response to sorbitol (500 mM) or SR59230A (10 µM) in the presence or absence of 8-Br-cAMP treatment (1 mM, 30 min). Values represent means ± S.E.M. (n = 6, performed in duplicate; ***, P < 0.001; **, P < 0.01). The immunoblot is representative of six experiments performed in duplicate. 8-Br-cAMP did not affect basal p38 MAPK phosphorylation (104 ± 14.2%). Sorbitol and SR59230A increased p38 MAPK phosphorylation (710 ± 71.2 and 294 ± 32.0%, respectively), and the responses were significantly inhibited (to 198 ± 38.7 and 111 ± 13.7%, respectively) in the presence of 8-Br-cAMP.

View larger version (42K): [in this window] [in a new window]   Fig. 7. Western blots of p38 MAPK phosphorylation in response to CL316243 and SR59230A in CHO-K1 cells expressing mouse 3-AR. Concentration-response curves for p38 MAPK phosphorylation in response to 15-min exposure to CL316243 or SR59230A in cells expressing 3-AR at high or low levels, with, at the right, representative immunoblots from six experiments performed in duplicate (P-p38 MAPK, phosphorylated p38 MAPK; T-p38 MAPK, total p38 MAPK). Each point represents the mean ± S.E.M. (n = 6, performed in duplicate). In CHO3H cells, the maximum responses of phospho/total p38 MAPK elicited by CL316243 and SR59230A over basal were 120 ± 4 and 979 ± 79%, respectively. In CHO3L cells, the maximum responses to CL316243 and SR59230A were 400 ± 25 and 1162 ± 69%, respectively.

Examination of the Effect of CL316243 [GenBank] and SR59230A on Erk1/2 by Western Blotting. Because recent studies have demonstrated that a number of ligands acting as inverse agonists at the ₁and ₂-AR for cAMP accumulation display the properties of agonists, neutral antagonists, or inverse agonists for Erk1/2 activation (Azzi et al., 2003; Baker et al., 2003; Galandrin and Bouvier, 2006), we also examined the effects of CL316243 [GenBank] and SR59230A on Erk1/2 phosphorylation in CHO-K1 cells expressing ₃-AR at high and low levels. We found that in contrast to p38 MAPK phosphorylation, the level of Erk1/2 phosphorylation caused by activation of the ₃-AR was similar in lowand high-expressing cells, but as with p38 MAPK phosphorylation, the response to SR59230A was greater than the response to CL316243 [GenBank] (Fig. 8). In CHO₃H cells, the maximum responses expressed as phospho/total Erk1/2 ratio elicited by CL316243 [GenBank] and SR59230A over basal were 341 and 775%, respectively (Fig. 8 and Table 1). In CHO₃L cells, CL316243 [GenBank] and SR59230A caused similar phospho/total Erk1/2 responses to CHO₃H cells, with maximum responses of 384 and 886%, respectively (Fig. 8). CL316243 [GenBank] and SR59230A had comparable potency to each other and between the two cell lines (Table 1). The similarity between Erk1/2 phosphorylation in highand low-expressing cells was reflected in the abundance of total Erk1/2 protein; this was 3280 ± 210 (6) densitometric units in CHO₃H cells versus 3110 ± 470 (6) in CHO₃L cells. In both cell lines, therefore, the relative efficacy of CL316243 [GenBank] and SR59230A for Erk1/2 phosphorylation was again a clear reversal of that seen for cAMP accumulation.

View larger version (32K): [in this window] [in a new window]   Fig. 8. Western blots of Erk1/2 phosphorylation in response to CL316243 and SR59230A in CHO-K1 cells expressing mouse 3-AR. Concentration-response curves for Erk1/2 phosphorylation in response to 15-min exposure to CL316243 or SR59230A in cells expressing 3-AR at high or low levels, with, at the right, representative immunoblots from six experiments performed in duplicate (P-Erk1/2, phosphorylated Erk1/2; T-Erk1/2, total Erk1/2). Each point represents the mean ± S.E.M. (n = 6, performed in duplicate). In CHO3H cells, the maximum responses expressed as phospho/total Erk1/2 ratio elicited by CL316243 and SR59230A over basal were 341 ± 28 and 775 ± 68%, respectively. In CHO3L cells, the maximum responses to CL316243 and SR59230A were 384 ± 36 and 886 ± 45%, respectively.

Discussion
CL316243 and SR59230A display reversed orders of efficacy for cAMP compared with Erk1/2 and p38 MAPK signaling in CHO-K1 cells stably expressing the mouse ₃-AR. In CHO₃H cells, CL316243 [GenBank] and (–)-isoproterenol stimulate cAMP accumulation with similar efficacy, but CL316243 [GenBank] is 100-fold more potent and SR59230A is a partial agonist with potency equivalent to that of (–)-isoproterenol. In CHO₃L cells, (–)-isoproterenol and CL316243 [GenBank] have reduced potency, but the cAMP response to SR59230A is lost altogether, and the compound acts as a competitive antagonist. The situation is reversed with MAPK activation in response to CL316243 [GenBank] and SR59230A. In both highand low-expressing cells, SR59230A stimulates Erk1/2 phosphorylation with a higher efficacy than CL316243 [GenBank] and has an equivalent potency. This suggests that, in contrast to CL316243 [GenBank] , the conformation of the ₃-AR recognized or induced by SR59230A is more efficiently coupled to Erk1/2 signaling than to the cAMP pathway. This interpretation requires that pathways that diverge at the receptor level mediate Erk1/2 phosphorylation and cAMP signaling. Previous studies show that ₃-AR agonists increase Erk1/2 phosphorylation in cells expressing endogenous or transfected ₃-AR (Soeder et al., 1999; Hutchinson et al., 2002). Activation of Erk1/2 involves recruitment of c-Src in adipocytes (Cao et al., 2000; Lindquist et al., 2000) and CHO₃-cells (Hutchinson et al., 2002). H-89, forskolin, or cholera toxin do not affect Erk1/2 phosphorylation in CHO₃-cells (Hutchinson et al., 2002), suggesting that the G_s/adenylate cyclase/cAMP pathway is not involved (Gerhardt et al., 1999). Likewise, ₃-AR-mediated Erk1/2 phosphorylation is independent of G_i, because the response is insensitive to pertussis toxin, and _3a-AR do not couple to G_i (Hutchinson et al., 2002; Sato et al., 2005).

p38 MAPK phosphorylation in response to ₃-AR activation occurs in 3T3-L1 adipocytes to BRL37344, CGP12177A, and SR58611A, and, to a lesser extent, SR59230A (Mizuno et al., 2002), and in these cells and primary brown adipocytes (Cao et al., 2001, 2004), the G_s/adenylate cyclase/cAMP pathway is necessary. We show here in CHO₃ cells that p38 MAPK phosphorylation to SR59230A is substantially higher than to CL316243 [GenBank] . Although similar to Erk1/2 in terms of the relative efficacy displayed by CL316243 [GenBank] and SR59230A, the p38 MAPK data show differences in drug potency and efficacy between CHO₃L and CHO₃H cells. In particular, SR59230A has higher efficacy for p38 MAPK phosphorylation in CHO₃L than in CHO₃H cells and a 30-fold higher potency in CHO₃L cells. CL316243 [GenBank] produces modest p38 MAPK phosphorylation in CHO₃L cells but relatively little in CHO₃H cells. These data suggested that there is an inverse relationship between cAMP and p38 MAPK signaling in CHO₃ cells. Maximal p38 MAPK activation is produced by SR59230A in CHO₃L cells, in which it produces little (Hutchinson et al., 2005) or no (present study) cAMP accumulation. In CHO₃H cells, SR59230A is a partial agonist for cAMP production, and efficacy and potency for p38 MAPK activation are lower. Likewise, CL316243 [GenBank] produced some p38 MAPK activation in CHO₃L cells but very little in CHO₃H cells in which it powerfully activates cAMP accumulation. The relationship between receptor expression levels and the altered p38 MAPK response therefore probably reflects the greater ability of CHO₃H cells to generate cAMP. This conclusion is supported by the finding that 8-Br-cAMP did not increase p38 MAPK phosphorylation but inhibited responses to both SR59230A and sorbitol.

The starting point for the present study was that in CHO₃L and 3T3-F442A cells, the ₃-AR ligand SR59230A is an agonist for ECAR but a competitive antagonist for cAMP responses (Hutchinson et al., 2005), suggesting that the ECAR response is mediated by pathway(s) other than cAMP. Here, we show that ECAR responses to (–)-isoproterenol, CL316243 [GenBank] , and SR59230A in CHO₃L cells were unaffected by inhibitors of adenylate cyclase, PKA, PI3K, Src, or MEK but markedly inhibited by the p38 MAPK inhibitor RWJ67657. The evidence strongly suggests that p38 MAPK is a major pathway used in the ECAR response to -AR ligands and is the sole pathway activated by SR59230A. This is consistent with the observation that pEC₅₀ values for SR59230A-stimulated ECAR and p38 MAPK phosphorylation are equivalent (6.0 and 6.2, respectively; Table 1). In CHO₃H cells, in contrast, ECAR responses to (–)-isoproterenol, CL316243 [GenBank] , or SR59230A were inhibited by DDA, H-89, and only partially inhibited by LY294002, PP2, or RWJ67657. Thus, the ECAR response in CHO₃H cells is predominantly mediated by the cAMP cascade, whereas PI3K, Src, and p38 MAPK have minor roles. This is supported by the observation that in CHO₃H cells, pEC₅₀ values for ECAR and cAMP accumulation are similar for CL316243 [GenBank] (10.6 and 9.8, Table 1) and SR59230A (7.8 and 7.9, Table 1). For SR59230A, the inhibitor studies (Figs. 3 and 4) and the pEC₅₀ values (Table 1) suggest a link between the cAMP and ECAR responses in CHO₃H cells and between p38 MAPK and ECAR in CHO₃L cells.

The concept of ligand-directed signaling is a topic of immense interest to pharmacologists and has been explained in terms of the ability of ligands to form distinct conformational complexes with the receptor (Kenakin, 2003; Urban et al., 2007). Recent studies have provided evidence that structurally distinct ligands differentially interact with basal state conformations of the ₂-AR to produce distinct conformational states, resulting in qualitatively different responses (Swaminath et al., 2005). Although the interaction of CL316243 [GenBank] and SR59230A with different conformational states of the ₃-AR may explain the differences in ability to activate cAMP accumulation and Erk1/2 phosphorylation, there is an additional layer of complexity imposed by the interaction between the cAMP and p38 MAPK signaling pathways that further accentuates the differences in pharmacological profile. Specifically, the pEC₅₀ values for the four functional bioassays and binding affinities determined in previous studies are compared in Table 1. As expected, the pEC₅₀ values for the recognized agonists (–)-isoproterenol and CL316243 [GenBank] in all four bioassays are higher than the binding affinities. The pEC₅₀ values for SR59230A are much closer to binding affinities except for the p38 MAPK and ECAR (downstream of p38 MAPK) assays. There is an inverse relationship between the magnitude of the cAMP response to SR59230A and the pEC₅₀ value for the p38 MAPK response. In CHO₃H cells in which SR59230A produced a substantial cAMP response, the pEC₅₀ for p38 MAPK is lower than the pK_i value from binding, whereas in CHO₃L cells, in which SR59230A produced little or no cAMP, the difference is less. To have this effect, classic receptor theory suggests that generation of cAMP must be associated with a lower affinity state of the ₃-AR for SR59230A, but the mechanism involved is unclear. However, PKA phosphorylation of the receptor cannot provide an explanation, because the mouse _3a-AR contains no PKA phosphorylation sites.

Several recent studies describe activation of Erk1/2 phosphorylation by drugs classified as -AR antagonists in cells expressing ₁or ₂-AR (Azzi et al., 2003; Baker et al., 2003). In particular, Galandrin and Bouvier (2006) demonstrated that a wide range of -AR ligands have complex efficacy profiles for cAMP generation and Erk1/2 activation at both ₁and ₂-ARs. Receptor-dependent activation of signaling pathways clearly depends on the array and abundance of signaling proteins present in a given cell type. We have shown that ₃-AR-mediated Erk1/2 phosphorylation displays similar properties in cells with high or low receptor abundance. In contrast, p38 MAPK responses are influenced by the level of receptor expression due to the interaction between cAMP and p38 MAPK signaling. Our demonstration of Erk1/2 and p38 MAPK signaling to SR59230A in cells expressing physiological receptor levels increases the relevance of this study to cells endogenously expressing receptors.

Given the importance of -AR antagonists in the treatment of cardiac failure, it will be important to determine whether p38 MAPK and Erk1/2 activation in response to -AR ligands occurs in tissues natively expressing -ARs. p38 MAPK have important roles in cellular responses to external stress signals, such as cell growth and inflammation. Long-term overexpression of p38 MAPK in rat cardiac tissues causes cell proliferation, inflammation, and fibrosis (Tenhunen et al., 2006a), yet short-term rescue of p38 MAPK after myocardial infarction protects by decreasing left ventricular remodelling and fibrosis and enhancing angiogenesis (Tenhunen et al., 2006b). Activation of p38 MAPK by -AR antagonists could influence a number of cardiac cell types, including myocytes, fibroblasts, and vascular endothelial cells that differentially express the three -ARs.

	Footnotes

 
This work was supported by the National Health and Medical Research Council (NHMRC) of Australia Project grant 236884 (to R.J.S.). D.S.H. is an NHMRC CJ Martin Fellow, M.S. is a Monash University Research Scholar.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.107.035337.

ABBREVIATIONS: AR adrenoceptor; CHO Chinese hamster ovary; ECAR, extracellular acidification rate; PI3K, phosphoinositide 3-kinase; MAPK, mitogen-activated protein kinase; Erk1/2, extracellular signal-regulated protein kinase 1/2; CYP, cyanopindolol; SR59230A, 3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate; CL316243 [GenBank] , (R, R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl] 1,3-benzodioxole-2,2-decarboxylate; RWJ67657, 4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine; PD98059, 2'-amino-3'-methoxyflavone; DDA, 2', 3'-dideoxyadenosine; H-89, N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride; PKA, protein kinase A; IBMX, 3-isobutyl-1-methylxanthine; BSA, bovine serum albumin; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; MEK, mitogen-activated protein kinase kinase; 8-Br-cAMP, 8-bromoadenosine 3', 5'-cAMP; BRL37344, (4-(2-((2-(3-chlorophenyl)-2-hydroxyethyl) amino) propyl) phenoxy) acetic acid; CGP12177A, 4-(3-tert-butylamino-2-hydroxypropoxy) benzimidazol-2-one; SR58611A, N-(7-hydroxy-1,2,3,4-tetrahydronaphth-2-yl)-2-hydroxy-2-(3-chlorophenyl) ethanol; ICI118551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl) oxy]-3-[(1-methylethyl) amino]-2-butanol; CHO₃H, ₃ adrenoceptor expressed in Chinese hamster ovary cells at high levels; CHO₃L, ₃ adrenoceptor expressed in Chinese hamster ovary cells at low levels.

¹ Current affiliation: Department of Cellular and Molecular Pharmacology, Hokkaido University Graduate School of Medicine, Hokkaido, Japan.

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

	References

Top Abstract References

 
Azzi M, Charest PG, Angers S, Rousseau G, Kohout T, Bouvier M, and Pineyro G (2003) -Arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors. Proc Natl Acad Sci USA 100: 11406–11411.[Abstract/Free Full Text]

Baker JG, Hall IP, and Hill SJ (2003) Agonist and inverse agonist actions of -blockers at the human ₂-adrenoceptor provide evidence for agonist-directed signaling. Mol Pharmacol 64: 1357–1369.[Abstract/Free Full Text]

Brahmadevara N, Shaw AM, and MacDonald A (2003) Evidence against ₃-adrenoceptors or low affinity state of ₁-adrenoceptors mediating relaxation in rat isolated aorta. Br J Pharmacol 138: 99–106.[CrossRef][Medline]

Candelore MR, Deng L, Tota L, Guan XM, Amend A, Liu Y, Newbold R, Cascieri MA, and Weber AE (1999) Potent and selective human ₃-adrenergic receptor antagonists. J Pharmacol Exp Ther 290: 649–655.[Abstract/Free Full Text]

Cao W, Daniel KW, Robidoux J, Puigserver P, Medvedev AV, Bai X, Floering LM, Spiegelman BM, and Collins S (2004) p38 mitogen-activated protein kinase is the central regulator of cyclic AMP-dependent transcription of the brown fat uncoupling protein 1 gene. Mol Cell Biol 24: 3057–3067.[Abstract/Free Full Text]

Cao W, Luttrell LM, Medvedev AV, Pierce KL, Daniel KW, Dixon TM, Lefkowitz RJ, and Collins S (2000) Direct binding of activated c-Src to the ₃-adrenergic receptor is required for MAP kinase activation. J Biol Chem 275: 38131–38134.[Abstract/Free Full Text]

Cao W, Medvedev AV, Daniel KW, and Collins S (2001) -Adrenergic activation of p38 MAP kinase in adipocytes: cAMP induction of the uncoupling protein 1 (UCP1) gene requires p38 MAP kinase. J Biol Chem 276: 27077–27082.[Abstract/Free Full Text]

Clarke WP (2005) What's for lunch at the conformational cafeteria? Mol Pharmacol 67: 1819–1821.[Abstract/Free Full Text]

Dumas M, Dumas JP, Bardou M, Rochette L, Advenier C, and Giudicelli JF (1998) Influence of -adrenoceptor agonists on the pulmonary circulation. Effects of a ₃-adrenoceptor antagonist, SR59230A. Eur J Pharmacol 348: 223–228.[CrossRef][Medline]

Fredriksson JM, Thonberg H, Ohlson KB, Ohba K, Cannon B, and Nedergaard J (2001) Analysis of inhibition by H89 of UCP1 gene expression and thermogenesis indicates protein kinase A mediation of ₃-adrenergic signalling rather than ₃-adrenoceptor antagonism by H89. Biochim Biophys Acta 1538: 206–217.[Medline]

Galandrin S and Bouvier M (2006) Distinct signaling profiles of ₁and ₂-adrenergic receptor ligands toward adenylyl cyclase and mitogen-activated protein kinase reveals the pluridimensionality of efficacy. Mol Pharmacol 70: 1575–1584.[Abstract/Free Full Text]

Gerhardt CC, Gros J, Strosberg AD, and Issad T (1999) Stimulation of the extracellular signal-regulated kinase 1/2 pathway by human ₃ adrenergic receptor: new pharmacological profile and mechanism of activation. Mol Pharmacol 55: 255–262.[Abstract/Free Full Text]

Horinouchi T and Koike K (2001) Agonistic activity of SR59230A at atypical -adrenoceptors in guinea pig gastric fundus and duodenum. Eur J Pharmacol 416: 165–168.[CrossRef][Medline]

Hutchinson DS, Bengtsson T, Evans BA, and Summers RJ (2002) Mouse _3aand _3b-adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signaling pathways. Br J Pharmacol 135: 1903–1914.[CrossRef][Medline]

Hutchinson DS, Sato M, Evans BA, Christopoulos A, and Summers RJ (2005) 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]. J Pharmacol Exp Ther 312: 1064–1074.[Abstract/Free Full Text]

Kenakin TP (2003) The secret lives of GPCRs. Drug Discov Today 8: 674.[CrossRef][Medline]

Khaled AR, Moor AN, Li A, Kim K, Ferris DK, Muegge K, Fisher RJ, Fliegel L, and Durum SK (2001) Trophic factor withdrawal: p38 mitogen-activated protein kinase activates NHE1, which induces intracellular alkalinization. Mol Cell Biol 21: 7545–7557.[Abstract/Free Full Text]

Lindquist JM, Fredriksson JM, Rehnmark S, Cannon B, and Nedergaard J (2000) ₃and ₁-adrenergic Erk1/2 activation is Srcbut not G_i-mediated in Brown adipocytes. J Biol Chem 275: 22670–22677.[Abstract/Free Full Text]

Manara L, Badone D, Baroni M, Boccardi G, Cecchi R, Croci T, Giudice A, Guzzi U, Landi M, and Le Fur G. (1996) Functional identification of rat atypical -adrenoceptors by the first ₃-selective antagonists, aryloxypropanolamino tetralins. Br J Pharmacol 117: 435–442.[Medline]

Mizuno K, Kanda Y, Kuroki Y, Nishio M, and Watanabe Y (2002) Stimulation of ₃-adrenoceptors causes phosphorylation of p38 mitogen-activated protein kinase via a stimulatory G protein-dependent pathway in 3T3–L1 adipocytes. Br J Pharmacol 135: 951–960.[CrossRef][Medline]

Penn RB, Parent JL, Pronin AN, Panettieri RA Jr, and Benovic JL (1999) Pharmacological inhibition of protein kinases in intact cells: antagonism of -adrenergic receptor ligand binding by H-89 reveals limitations of usefulness. J Pharmacol Exp Ther 288: 428–437.[Abstract/Free Full Text]

Sato M, Hutchinson DS, Bengtsson T, Floren A, Langel U, Horinouchi T, Evans BA, and Summers RJ (2005) Functional domains of the mouse ₃-adrenoceptor associated with differential G protein coupling. J Pharmacol Exp Ther 315: 1354–1361.[Abstract/Free Full Text]

Soeder KJ, Snedden SK, Cao W, Della Rocca GJ, Daniel KW, Luttrell LM, and Collins S (1999) The ₃-adrenergic receptor activates mitogen-activated protein kinase in adipocytes through a Gi-dependent mechanism. J Biol Chem 274: 12017–12022.[Abstract/Free Full Text]

Strosberg AD and Pietri-Rouxel F (1997) The ₃-adrenoceptor constitutes indeed a versatile receptor. Trends Pharmacol Sci 18: 52–53.[Medline]

Swaminath G, Deupi X, Lee TW, Zhu W, Thian FS, Kobilka TS, and Kobilka B (2005) Probing the ₂-adrenoceptor binding site with catechol reveals differences in binding and activation by agonists and partial agonists. J Biol Chem 280: 22165–22171.[Abstract/Free Full Text]

Tenhunen O, Rysa J, Ilves M, Soini Y, Ruskoaho H, and Leskinen H (2006a) Identification of cell cycle regulatory and inflammatory genes as predominant targets of p38 mitogen-activated protein kinase in the heart. Circ Res 99(5): 485–493.[Abstract/Free Full Text]

Tenhunen O, Soini Y, Ilves M, Rysa J, Tuukkanen J, Serpi R, Pennanen H, Ruskoaho H, and Leskinen H (2006b) p38 Kinase rescues failing myocardium after myocardial infarction: evidence for angiogenic and anti-apoptotic mechanisms. FASEB J 20: 1907–1909.[Abstract/Free Full Text]

Urban JD, Clarke WP, von Zastrow M, Nichols DE, Kobilka B, Weinstein H, Javitch JA, Roth BL, Christopoulos A, Sexton PM, et al. (2007) Functional selectivity and classical concepts of quantitative pharmacology. J Pharmacol Exp Ther 320: 1–13.[Abstract/Free Full Text]

Wadsworth SA, Cavender DE, Beers SA, Lalan P, Schafer PH, Malloy EA, Wu W, Fahmy B, Olini GC, Davis JE, et al. (1999) RWJ 67657, a potent, orally active inhibitor of p38 mitogen-activated protein kinase. J Pharmacol Exp Ther 291: 680–687.[Abstract/Free Full Text]

Related articles in MolPharm:

Ligand-Directed Signaling: 50 Ways to Find a Lover

Martin C. Michel and Astrid E. Alewijnse
MolPharm 2007 72: 1097-1099. [Abstract] [Full Text]  

This article has been cited by other articles:

				M. Sato, D. S. Hutchinson, B. A. Evans, and R. J. Summers Mol. Pharmacol., November 1, 2008; 74(5): 1417 - 1428. [Abstract] [Full Text] [PDF]

				M. C. Michel and A. E. Alewijnse Ligand-Directed Signaling: 50 Ways to Find a Lover Mol. Pharmacol., November 1, 2007; 72(5): 1097 - 1099. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: mol.107.035337v1 72/5/1359    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Related articles in MolPharm Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sato, M. Articles by Summers, R. J. Search for Related Content PubMed PubMed Citation Articles by Sato, M. Articles by Summers, R. J.