J. Anim. Sci. 2003. 81:122-129
© 2003 American Society of Animal Science
Stereoselectivity of porcine ß-adrenergic receptors for ractopamine stereoisomers1,2
S. E. Mills*,3,
J. Kissel*,
C. A. Bidwell* and
D. J. Smith
* Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 and
and
USDA, ARS, Biosciences Research Laboratory, Fargo, ND 58105
3 Correspondence:
Lilly Hall (phone 765-494-4845; fax 765-494-9346; E-mail:
smills{at}purdue.edu).
 |
Abstract
|
|---|
Ractopamine HCl is a ß-adrenergic receptor (ßAR) ligand approved for use in swine to enhance carcass leanness. Ractopamine is produced commercially as a mixture of four stereoisomers (RR, RS, SR, SS). In order to determine which stereoisomers are active in the pig and whether they exhibit ßAR subtype selectivity, receptor affinity and adenylyl cyclase activation were determined using cloned porcine ß1- and ß2AR expressed in Chinese hamster ovary (CHO) cells. Dissociation constants (Kd) were determined by competitive displacement of [125I]iodocyanopindolol binding by ractopamine stereoisomers. The RR isomer had the highest affinity for both ß1- and ß2AR (Kd of 29 and 26 nM, respectively). Dissociation constants for the other stereoisomers were higher (RS = 463 and 78 nM, SR = 3,230 and 831 nM, SS = 16,600 and 3,530 nM for the ß1- and ß2AR, respectively) relative to the RR stereoisomer. Isoproterenol stimulated adenylyl cyclase activity 600% relative to basal rates in CHO cells, regardless of ßAR subtype. Ractopamine stereoisomers did not significantly (P > 0.05) stimulate adenylyl cyclase through the ß1AR at moderate (near Kd) or high (10-4 M) concentrations. In contrast, the RR isomer increased adenylyl cyclase activity 200 to 300% relative to basal rates through the ß2AR at moderate and high concentrations; the SR stereoisomer increased adenylyl cyclase activity nearly 100%. Neither the RS nor SS stereoisomers were effective in activating adenylyl cyclase activity through the ß2AR. A pattern of stereoselective activation similar to that for adenylyl cyclase also was exhibited for lipolysis using porcine adipocytes. The RR stereoisomer was equal to isoproterenol in stimulating lipolysis, whereas the SR isomer was 50% as effective; the RS and SR stereoisomers did not stimulate lipolysis in porcine adipocytes. The porcine ßAR exhibited stereoselectivity toward ractopamine stereoisomers with the RR isomer exhibiting the highest affinity for the ß1- and ß2AR. In contrast, ractopamine stereoisomers seemed to be more effective at eliciting adenosine cyclic 3',5'-phosphate responses from ß2AR than ß1AR. The RR isomer is likely the functional stereoisomer of ractopamine, but its effectiveness may be compromised by the presence of competing isomers, in particular the RS stereoisomer.
Key Words: ß-Adrenergic Receptor • Growth • Pigs
|
Introduction
|
|---|
Ractopamine is a phenethanolamine ß-adrenergic receptor (ßAR) agonist that is commercially available for enhanced muscle protein growth in pigs. The ßAR belongs to a family of seven transmembrane domain proteins that are coupled to stimulatory G-proteins. Three ßAR subtypes have been cloned, and evidence for a fourth subtype has been reported (Kaumann, 1997; Tate et al., 1991). Ractopamine is reported to be selective for the ß1AR (Smith et al., 1990; Moody et al., 2000). Ractopamine’s reported ß1AR-selectivity contrasts the ß2AR-selectivity reported for other ß-agonists effective at growth modification in rodents such as clenbuterol (Cohen et al., 1982; Orcutt et al., 1989), cimaterol (Brittain et al., 1976; Byrem et al., 1998), salbutamol (Colbert et al., 1991), and L644,969 (Convey et al., 1987). In rodents, ß1AR-selective agonists did not increase growth (Convey et al., 1987; Emery et al., 1984), indicating a unique role for ß2AR ligands in rodents. If ractopamine targets a different ßAR subtype, then the mechanism regulating muscle growth may also differ (Moody et al., 2000). For the pig, however, there is no evidence that ractopamine exhibits selectivity for the mixed population of ßAR in pig tissues (Spurlock et al., 1993) or for cloned ßAR (Cao, 1998; Liang et al., 2001). However, the true kinetics of ractopamine binding may be confounded by the presence of multiple stereoisomers.
The commercial preparation of ractopamine contains four stereoisomers resulting from the presence of two chiral carbons (Figure 1
). Ricke et al. (1999) point out that because ractopamine is a racemic mixture, not all isomers may be biologically active and the true kinetics of the active isomer may be modified by the presence of competing isomers. Therefore, it was of interest to determine which stereoisomers bind and activate the cloned porcine ßAR and whether subtype selectivity is exhibited.
View larger version (7K):
[in this window]
[in a new window]
|
Figure 1. Structure of ractopamine HCl. Chiral carbons are denoted by asterisks; the hydroxyl substitution of the chiral carbon in the position ß to the aliphatic nitrogen is common to the natural catecholamines. Ractopamine is an equilmolar mixture of four stereoisomers.
|
|
|
Materials and Methods
|
|---|
Materials.
Porcine ß1AR and ß2AR (pßAR) have been cloned and expressed in stable cell lines (Cao, 1998; Liang et al., 2000). Chinese hamster ovary cells do not express endogenous ßAR and the expressed genes generate pure forms of the porcine ßAR. Ractopamine {(1 R *, 3 R * ), (1 R *, 3 S* ) - 4 - hydroxy - 
- [[[3 - (4 - h y -droxy [ 14C] phenyl) - 1 - methylpropyl] amino] methyl] -benzenemethanol hydrochloride} stereoisomers were synthesized as described by Ricke et al. (1999). The stereochemical purity of each isomer was: RR: 99.0% RR, 0.9% SR, 0.1% SS, 0.1% RS; RS: 0.1% RR, 0.1% SR, 0.5% SS, 99.4% RS; SR: 0.5% RR, 99.5% SR, 0% SS and RS; and SS: 0.1% RR, 0% SR, 98.4% SS and 1.6% RS. (–)Isoproterenol was purchased from Sigma Chemical (St. Louis, MO). The radioligand [125I]iodocyanopindolol ([125I]CYP]) was from Amersham (2,000 Ci/mmol, Arlington Heights, IL). Culture media F12 and Dulbecco’s modified Eagle’s medium (DMEM) were from Gibco-BRL (Rockville, MD) and other reagents were from Sigma Chemical (St. Louis, MO) unless otherwise specified.
Cell Culture.
Stably transfected Chinese hamster ovary (CHO) cell lines expressing either pß1AR or pß2AR were grown in an atmosphere of 95% air and 5% CO2 at 37°C in a 1:1 F12:DMEM media containing 100 U/mL of ampicillin, 200 U/mL of penicillin, 200 µg/mL of streptomycin, 10-8 M Se, and 1.2 mg/mL of NaHCO3 in 1.5 mM HEPES, pH 7.4, plus 10% fetal bovine serum and G418 (0.5 mg/mL). Confluent CHO cells were washed twice with ice-cold PBS (pH 7.4), scraped into 7 mL of hypotonic lysing buffer (5 mM Tris-HCl, 5 mM EDTA, pH 7.4, 2 µM leupeptin, and 2 µM pepstatin), and homogenized with a Wheaton glass tissue grinder. Homogenates were centrifuged at 48,000 x g for 20 min at 4°C. Membrane pellets were suspended in incubation buffer (50 mM Tris-HCl, 5 mM MgCl2, 1 mM EDTA, pH 7.4, 2 µM leupeptin, and 2 µM pepstatin) and centrifuged as above. The final pellets were suspended in incubation buffer and frozen in liquid nitrogen.
Radioligand Binding.
Membranes (5 to 10 µg) from cells were incubated in 17 x 100 mm polyethylene tubes in the presence of [125I]CYP and test ligands in a final volume of 0.15 mL. Duplicate tubes were shaken in a gyratory shaker at 37°C for 60 min followed by filtration through Whatman GF/C glass fiber filters and were washed with 10 mL of ice-cold incubation buffer. Radioactivity was quantified in a Packard Cobra 
-counter (Meriden, CT). Nonspecific binding was defined as [125I]CYP bound in the presence of 100 µM (–)-isoproterenol. Specific binding was calculated as the differences between total binding and nonspecific binding. Binding parameters and the best-fit models were determined using nonlinear regression analysis (Prism, GraphPad Software Inc., San Diego, CA).
Adenylyl Cyclase.
Assays were performed in 1.5-mL microcentrifuge tubes in a total volume of 0.15 mL at 37°C for 10 min. Final concentrations of assay components were 25 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mM GTP, 1 mM dithiothreitol, 25 mM phosphocreatine, 3 U/mL of creatinephosphokinase, 0.1% BSA, 0.5 mM ATP, and 1 mM theophylline. Assay cocktail and ligands in a total volume of 0.063 mL were prewarmed for 5 min at 37°C. Reactions were initiated by adding cold membranes from CHO cell lines (0.087 mL) at 30-s intervals and shaking gently. Reactions were stopped after 10 min by adding 0.5 mL of cold 8 mM EDTA. Following incubation, assay tubes were centrifuged for 10 min at 4°C at 10,000 x g and the supernatants stored at -20°C. The adenosine cyclic 3',5'-phosphate (cAMP) was assayed by RIA using the protocol supplied with the cAMP antibody (Calbiochem, San Diego, CA). The second antibody was from Qiagen (Bio-Mag goat anti-rabbit IgG; Valencia, CA). Samples and standards were acetylated to increase the sensitivity of the assay. The assay detected as little as 2 fmol of cAMP and basal and isoproterenol-stimulated rates were approximately 4 and 50 pmol•min-1•mg of protein-1, respectively.
Adipocyte Preparation and Incubation.
The middle layer of subcutaneous adipose tissue was removed from the back (8th to 12th rib) of market-weight York x Landrace hogs at the Purdue University abattoir. Hogs were killed by exsanguination following electrical stunning between 0800 and 1000 h. Adipose tissue was transported to the laboratory in 37°C buffered saline (0.15 M NaCl, - 1 mM HEPES, pH 7.4). Adipocytes were isolated by collagenase digestion and cell number was determined from the average cell size as measured from osmium tetroxide fixed cells and total lipid (Liu et al., 1989). Adipocytes (approximately 105 cells/mL) were washed and suspended in incubation buffer (Krebs-Ringer bicarbonate containing 1.25 mM CaCl2, 0.5 mM ascorbic acid, 10 mM HEPES, 5 mM glucose, and 3% BSA, pH 7.4, as described by Liu et al. (1989). To quantify rates of lipolysis, duplicate 0.5-mL aliquots of the cell suspension were incubated in 17 x 100 mm polyethylene tubes in an atmosphere of 5% CO2 in oxygen. Tubes contained theophylline (0.4 mM) and the test ßAR ligands isoproterenol or ractopamine stereoisomers. Vials were shaken in a gyratory water bath at 37°C for 2 h. Incubations were stopped by adding 0.025 mL of 35% HCLO4, and glycerol was quantified in neutralized, protein-free extracts using a commercial kit adapted for 96-well plates (GPO-Trinder triglyceride kit; Sigma Chemical Co.). Lipolytic rates were expressed as nmoles glycerol released•min-1•106 cells-1.
Data were analyzed using the GLM procedures of SAS (SAS Inst. Inc., Cary, NC). Three pigs were used for lipolysis studies, whereas receptor binding and adenylyl cyclase assays were conducted with pooled membranes from cultured cells. Activation of lipolysis or adenylyl cyclase was detected using single degree of freedom orthogonal contrasts for differences from basal activity.
|
Results
|
|---|
Affinities of the porcine ßAR for the ractopamine stereoisomers were determined by competitive displacement of [125I]CYP from cloned ß1AR and ß2AR (Figure 2). All curves best fit a one-site model using nonlinear regression except for the curve generated by the displacement of the RR stereoisomer from the ß2AR, which best fit a two-site model (data not shown). Biphasic binding is characteristic of agonist interaction with the ßAR and represents binding of the agonist to different conformational states of the receptor (Samama et al., 1993). Antagonists do not exhibit biphasic binding (Liang and Mills, 2001). Because biphasic-binding curves may interfere with estimates of binding affinity, GTP may be added to incubations to convert the ßAR to the low affinity state and a single class of binding sites (Samama et al., 1993). Therefore, for RR at the ß2AR, binding experiments were conducted in the presence of 100 µM guanosine 5-triphosphate (GTP). Dissociation curves generated with the RR stereoisomer in the presence of GTP modeled to one site (Figure 2), and Kd values were similar to estimates determined using data generated without GTP and the data fit to a one-site model (data without GTP are not shown). The presence of GTP does not affect binding of ligands that model to one site (Liang and Mills, 2001) and therefore, GTP was not included for the other isomers. Affinity estimates for the isomers were calculated using Kd values for [125I]CYP of 19 and 14.5 pM for ß1AR and ß2AR, respectively (Cao, 1998; Liang et al., 2000), and results are shown in Table 1. The RR isomer exhibited the highest affinity of all stereoisomers for both receptor subtypes, but the affinity values for the RR stereoisomer were not different (P > 0.05) between ß1AR and ß2AR. The orientation of the hydroxyl group around the ß-carbon (relative to the aliphatic nitrogen) had the greatest impact on affinity measures because the RR and RS isomers had greater affinities than the SR and SS isomers. The orientation around the asymmetric carbon adjacent to the aliphatic nitrogen (Figure 1
) also affected binding, with the R orientation being the most favorable. The SS isomer had the lowest affinity for each subtype and the rank order was similar for both subtypes (RR > RS 
SR > SS). Except for the RR isomer, the ractopamine isomers had greater affinities for ß2AR than the ß1AR. The mix of isomers (equal mix of all four isomers) was evaluated for the ß2AR and the Ki fell between the RR and RS, likely reflecting the primary titration by these two isomers and the proportionately reduced concentration of each isomer in the mixture.
View larger version (19K):
[in this window]
[in a new window]
|
Figure 2. Competitive displacement of specific [125I]iodocyanopindolol binding by ractopamine stereoisomers from the porcine ß1- and ß2-adrenergic receptors in membranes from Chinese hamster ovary cells. Membranes were incubated with ~60 pM [125I]CYP at 37°C for 60 min in the presence of multiple concentrations of the competitors. The kinetics of the RR stereoisomer binding to the ß2AR was biphasic, and therefore incubations were repeated in the presence of 100 µM Gpp(NH)p, a guanosine 5'-triphosphate (GTP) analog. The data for (+)-GTP are shown for the RR stereoisomer. Data are expressed as the percentage of [125I]iodocyanopindolol ([125I]CYP) bound in the absence of competitor. Curves were fit by nonlinear regression and results are summarized in Table 1. Results are means of two to four independent experiments. Error bars were omitted for clarity.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1. Estimates of binding affinity of porcine ß-adrenergic receptors expressed in Chinese hamster ovary cells for ractopamine isomersa
|
|
The ability of the individual ractopamine stereoisomers to activate adenylyl cyclase was determined from CHO cells (Figure 3). Two concentrations were used for each stereoisomer, one near the Kd (low dose) of each isomer and the other at 10-4 M (high dose). Isoproterenol increased adenylyl cyclase activity five- to sixfold in membranes of CHO cells expressing either the ß1AR or ß2AR. The response to ractopamine isomers was less than that of isoproterenol, consistent with ractopamine being a partial agonist toward pig ßAR. None of the isomers significantly (P > 0.05) increased adenylyl cyclase activity through the ß1AR, and in some instances, rates were significantly decreased from basal. At the ß2AR, the RR stereoisomer was the most efficacious, increasing adenylyl cyclase activity two- to fourfold at low and high concentrations, respectively. The SR isomer also increased adenylyl cyclase twofold, but neither the RS nor SS stereoisomers were stimulatory. The mixture of stereoisomers was less efficacious than the RR stereoisomer at activating adenylyl cyclase. This may suggest competition by other isomers (RS) limits the effectiveness of RR.
View larger version (16K):
[in this window]
[in a new window]
|
Figure 3. Activation of adenylyl cyclase by ractopamine isomers through the porcine ß1- and ß2-adrenergic receptor in membranes from Chinese hamster ovary cells. Membranes were incubated for 10 min in the presence or absence of indicated ligand and the quantity of adenosine cyclic 3',5'-phosphate (cAMP) determined. Two concentrations of each ractopamine stereoisomer were tested; one near the Kd for each drug (L) and one at 10-4 M (H). B = basal (no addition); I = 10-4 M isoproterenol; RR, RS, SR, and SS are the stereoisomers of ractopamine; and "MIX" is an equimolar mixture of each stereoisomer. Data are the means and SEM for three or four independent experiments and are expressed as a percentage of the response to isoproterenol. Asterisks denote significant (P < 0.05) deviation from basal rate for ractopamine isomers.
|
|
Collectively, data derived from the cloned receptors indicate that each ractopamine stereoisomer binds with a unique affinity to each ßAR subtype, but that signaling through adenylyl cyclase may be restricted to the RR and SR stereoisomers. In addition, signal transduction was more efficiently coupled through the ß2AR than the ß1AR in the cloned receptor model. To determine whether ractopamine stereoisomer specificity was exhibited in pig adipose tissue, we quantified the activation of lipolysis in adipocytes (Figure 4). The porcine adipocyte expresses three ßAR subtypes with the ß1AR representing approximately 75%, the ß2AR approximately 20%, and the ß3AR approximately 5% of the total ßAR (McNeel and Mersmann, 1999; Liang et al., 2002). The concentration of each stereoisomer tested was 10-5 M, which was greater than the Kd for all isomers except for SS at the ß1AR. The RR stereoisomer and (–)isoproterenol were equally efficacious and stimulated lipolysis approximately 30-fold over basal lipolysis. The SR isomer was also stimulatory, but was only about 50% as effective as RR stereoisomer. Neither the RS nor the SS stereoisomers activated lipolysis, which was consistent with data derived from adenylyl cyclase activation experiments.
View larger version (16K):
[in this window]
[in a new window]
|
Figure 4. Stimulation of lipolysis by ractopamine stereoisomers in porcine adipocytes. Adipocytes were incubated for 2 h in the presence or absence of indicated ligand (10-4 M) and the quantity of glycerol determined. B = basal rate (no addition); Iso = isoproterenol; RR, RS, SR, and SS are ractopamine stereoisomers. Data are the means and SEM for three independent experiments.
|
|
|
Discussion
|
|---|
Phenethanolamines represent a class of compounds that may bind to - and/or ß-adrenergic receptors. The phenethanolamine class includes the natural neurohormones epinephrine and norepinephrine, and synthetic ßAR agonists, such as isoproterenol, salbutamol, clenbuterol, L644,969, and ractopamine (Ruffolo, 1991; Smith, 1998), among hundreds of others. ß-Adrenergic agonists are optically active, possessing a chiral center at the ß-carbon relative to the aliphatic amine. In order to have specificity to the ßAR, the substitution at the ß-carbon must be a hydroxyl. ß-Adrenergic receptors generally exhibit a stereoselectivity of at least 100-fold in favor of levorotatory phenethanolamines (Ruffolo, 1991). The proper orientation of the ß-hydroxyl group appears to allow a three-point interaction of the aliphatic amine, ß-hydroxyl group, and phenyl group present on levorotatory stereoisomers with ßAR. A two-point interaction between the receptor and the aliphatic amine and phenyl group, but not the ß-hydroxyl group, is characteristic of the binding of dextrorotatory (S+) stereoisomers to ßAR (Ruffolo, 1991). For efficient signal transduction after binding, a three-point interaction is required. Using site-directed mutagenesis of human ß2AR clones, Wieland et al. (1996) have shown that the stereochemical requirements for agonist binding and signal transduction are dependent upon asparagine residue 293 of transmembrane helix VI. The naturally occurring catecholamines are levorotatory. The pig ßAR is stereoselective as demonstrated by the 10-fold higher affinity for (–)-isoproterenol relative to (+)-isoproterenol in adipocyte membranes (Mersmann and McNeel, 1992; Spurlock et al., 1993a).
Ractopamine HCl contains two chiral carbons (Figure 1) and exists as an equal mixture of the four resulting stereoisomers (RR, RS, SR, SS) in the commercial preparation (Colbert et al., 1991). In the present study, the RR isomer had the highest affinity for the pig ß1AR and ß2AR. The RR stereoisomer was also the most effective agonist for activation of adenylyl cyclase through cloned receptors expressed in CHO cells (this study), stimulation of lipolysis in porcine adipocytes (this study), and activation of adenylyl cyclase in mouse skeletal muscle cells (Shappell et al., 2000). It is likely, therefore, that the RR stereoisomer is the most active of the four compounds in the pig. The RR isomer has also been shown to be the most active stereoisomer in rodents. Yen et al. (1983) first demonstrated that the RR isomer acutely stimulated lipolysis in vivo. Subsequently, Ricke et al. (1999) administered individual isomers to rats by osmotic mini-pumps and found that the RR isomer accounted for essentially all of the growth response of the racemic mixture. That RR is the most efficacious isomer for growth in the rat and at the pig ßAR provides supportive evidence that the ßAR is mediating the growth response in animals. These data are also consistent with the stereospecific effects of the RR isomer of the ß2-adrenergic agonist formoterol on the smooth muscle relaxation in guinea pig trachea (Källström et al., 1996). Formoterol is a ß-agonist that is similar to ractopamine in that it has two chiral carbons located in analogous positions as the chiral carbons in ractopamine. The SS stereoisomer of formoterol did not have any activity towards smooth muscle relaxation (a ß2AR mediated event).
The RR isomer had similar (P > 0.05) affinities for the ß1AR and ß2AR (29 to 25 nM), and therefore would be classified as nonselective for these two subtypes in the pig based on binding. In contrast, activation of adenylyl cyclase was only effective through the ß2AR. In both ßAR subtypes the RR isomer was, at best, a partial agonist because it activated adenylyl cyclase to a lesser degree than did isoproterenol. In the case of the ß1AR, no activation was detected. We have previously demonstrated that ractopamine is only a partial agonist compared to isoproterenol (Mills and Spurlock, 1995), and, under specific conditions in vitro, ractopamine functions as an antagonist to isoproterenol-stimulated lipolysis (Liu et al., 1989). Therefore, it was not surprising that RR was less efficacious than isoproterenol in activating the ßAR and initiating a signaling response. The fact that we were unable to detect activation of adenylyl cyclase in the ß1AR does not mean that ractopamine does not signal through the ß1AR. In fact, we have demonstrated that the RR stereoisomer stimulates the accumulation of cAMP in intact CHO cells expressing the ß1AR (data not shown). What these data demonstrate is that adenylyl cyclase activation is more efficacious through the ß2AR than the ß1AR. That the RR stereoisomer was equivalent to (–)-isoproterenol in stimulating lipolysis in pig adipocytes reflects the fact that pig adipocytes have spare receptors and that maximal lipolysis is achieved at submaximal intracellular cAMP concentrations. It will be of interest to determine which ßAR is activated by the RR stereoisomer in vivo because the pig adipocyte expresses all three ßAR subtypes in an approximate ratio of 72:20:8 ß1:ß2:ß3 (McNeel and Mersmann, 1999; Liang et al., 2002), and because we have suggested that the ß1AR is the primary ßAR regulating lipolysis (Mills 2000).
The suggestion that ractopamine is a more effective ß2AR agonist in the pig is contrary to the conclusions of others (see review by Moody et al., 2000). Moody et al. (2000) proposed that the mechanism of ractopamine action on growth may differ from the mechanism(s) of other ßAR ligands (i.e., L-644,969 and clenbuterol) because ractopamine is ß1AR selective, whereas other leanness-enhancing ß-agonists are purported to be ß2AR selective. However, evidence for ß1AR-selectivity is scant. Whereas Smith et al. (1990) indicated that ractopamine exhibited 15-fold selectivity for the ß1AR in a rat glioma cell line, Colbert et al. (1991) tested ractopamine in rat and guinea pig tissues and did not demonstrate significant affinity differences for either receptor, but did demonstrate greater efficacy through the ß2AR than the ß1AR, similar to our findings for pig ßAR. For the pig, there is no evidence that ractopamine exhibits receptor subtype selectivity (Coutinho et al., 1992; Spurlock et al., 1993b), and the RR stereoisomer was not selective using cloned pig ßAR. In fact, ractopamine appears to activate adenylyl cyclase more efficiently through the ß2AR, although more work is needed to determine whether the apparent subtype differences are relevant. Quantifying the response to ractopamine in the presence of highly selective antagonists toward the pß1AR or pß2AR would help resolve this issue. It remains to be determined unequivocally whether ractopamine does function differently from other lean-enhancing ßAR agonists; if so, it is not because of a preferential activation of the ß1AR.
All four ractopamine stereoisomers bound to the ß1AR and ß2AR, but none was as effective as the RR stereoisomer at activating adenylyl cyclase and adipocyte lipolysis. The finding that the SR stereoisomer stimulated adenylyl cyclase and adipocyte lipolysis, but that the RS did not, is surprising given the known stereoselectivity of the ßAR. Yen et al. (1983) evaluated the lipolytic response to stereoisomers of a phenethanolamine ß-agonist that was structurally similar to ractopamine (a phenyl group replaced the phenol on the ethanolamine portion of the ractopamine). All four isomers were lipolytic in mouse adipose tissue and the RR and RS stereoisomers were the most efficacious followed by the SS and SR stereoisomers. The authors did not indicate the stereochemical purity of each of the isomers. It is possible that the effect of the SR stereoisomer could have been partially affected by the presence of the RR stereoisomer (0.5%). However, we believe this is unlikely because neither the RS nor SS stereoisomers were stimulatory yet they also contained a small percentage of the RR isomer (0.1%), and the calculated effective concentration of RR in each fraction (RS, SR, and SS) was less than the concentration required for activation.
Data presented in the present study indicate that only the RR and SR stereoisomers were stimulatory through the porcine ßAR and the RR was the most effective. To the extent that the RS, SR, and SS stereoisomers could compete with RR stereoisomer for ßAR binding, the net response to RR stereoisomer would be expected to be diminished. The affinities of the SR and SS stereoisomers were at least 30 times lower than the affinities of the RR stereoisomer for the ß1AR and ß2AR and would not be expected to compete with the RR isomer at equimolar concentrations. Similarly, the RS stereoisomer had an affitity for the ß1AR approximately 15 times lower than the RR stereoisomer. It would also not be expected to significantly interfere with receptor activation by the RR stereoisomer. At the ß2AR, however, the RS isomer had approximately one-third the affinity than RR stereoisomer; at equimolar concentrations, competitive displacement of RR would likely occur. Because the RS stereoisomer was not stimulatory, a reduced response to RR stereoisomer would be expected in the presence of the RS isomer. Competition at the receptor between the RS and RR stereoisomers may explain the diminished response to the mix of isomers at the ß2AR compared to RR. The effect of the mixture of isomers on lipolysis in isolated adipocytes was not measured, but the predominance of the ß1AR in porcine adipocytes may mask the interference at the ß2AR. These data suggest that the presence of competing isomers in the racemic mixture may limit the effectiveness of the ractopamine, particularly through the ß2AR. However, this assumes that each isomer is metabolized at the same rate and that they appear in equal concentrations in the blood. This assumption may not hold for ractopamine, because swine tissues retain (+)-clenbuterol to a greater extent than (–)-clenbuterol (Smith, 2000). In rats, the RR stereoisomer was not more effective than native ractopamine, but the relative affinities of the isomers for the rat ßAR have not been published (Ricke et al., 1999).
|
Implications
|
|---|
Activation of ß-adrenergic receptors is an effective means to augment muscle growth. The ß2 -adrenergic receptor appears to mediate the growth response in rodent, but this cannot be stated for the pig, in part because of the limited knowledge about the affinity of different ligands for porcine ß-adrenergic receptors. Commercial preparations of ractopamine are an equal mixture of four stereoisomers, and here we demonstrate that the RR isomer is likely the functional ligand because it has the highest affinity and greatest ability to elicit cellular responses after receptor binding. The RR isomer is not subtype-selective in the pig, but may couple more efficiently to adenylyl cyclase through the ß2- than the ß1-receptor. Because ractopamine appears to be nonselective in the pig, it seems likely that ractopamine shares a common mechanism of action with other ß-adrenergic receptor ligands. In addition, the beneficial effects of RR may be limited by the presence of competing isomers.
|
Footnotes
|
|---|
1 Journal paper No. 16805 of the Purdue University Agric. Res. Prog. 
2 Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable.
Received for publication July 15, 2002.
Accepted for publication September 9, 2002.
|
Literature Cited
|
|---|
Brittain, R. T., C. M. Dean, and D. Jack. 1976. Sympathomimetic bronchodilator drugs. Pharmacol. Ther. [B] 2:423–462.[Medline]
Byrem, T. M., D. H. Beermann, and T. F. Robinson. 1998. The ß-agonist cimaterol directly enhances chronic protein accretion in skeletal muscle. J. Anim. Sci. 76:988–998.[Abstract/Free Full Text]
Cao, H. 1998. Molecular cloning and binding kinetics of the porcine ß1-adrenergic receptor. M.S. Thesis, Purdue Univ., West Lafayette, IN.
Cohen, M. L., K. S. Wiley, and K. G. Bemis. 1982. Analysis of the ß1 and ß2 adrenoceptor interactions of the partial agonist, clenbuterol (NAB365), in the rat jugular vein and atria. Naunyn. Schmiedebergs Arch. Pharmacol. 320:145–151.[Medline]
Colbert, W. E., P. D. Williams, and G. D. Williams. 1991. ß-Adrenoceptor profile of ractopamine HCl in isolated smooth and cardiac muscle tissues of rat and guinea-pig. J. Pharm. Pharmacol. 43:844–847.[Medline]
Convey, E. M., E. Rickes, Y. T. Yang, M. A. McElligott, and G. Olson. 1987. Effects of beta-adrenergic agonist L-644,969 on growth performance, carcass merit and meat quality. Proc. Recip. Meat Conf. 40:47–55.
Coutinho, L. L., W. G. Bergen, R. A. Merkel, and C. K. Smith II. 1992. Quantitative characterization of ß-adrenergic receptor subtypes in porcine adipocytes. Comp. Biochem. Physiol. C. 101:481–485.
Emery, P. W., N. J. Rothwell, M. J. Stock, and P. D. Winter. 1984. Chronic effects of ß2-adrenergic agonists on body composition and protein synthesis in the rat. Biosci. Rep. 4:83–91.[Medline]
Kaumann, A. J. 1997. Four ß-adrenoceptor subtypes in the mammalian heart. Trends Pharmacol. Sci. 18:70–76.[Medline]
Liang, W., and S. Mills. 2001. Profile of ligand binding to the porcine ß2-adrenergic receptor. J. Anim. Sci. 79:877–883.[Abstract/Free Full Text]
Liang, W., C. A. Bidwell, P. R. Collodi, and S. E. Mills. 2000. Expression of the porcine ß2-adrenergic receptor in Chinese hamster ovary cells. J. Anim. Sci. 78:2329–2335.[Abstract/Free Full Text]
Liang, W., and S. E. Mills. 2002. Quantitative analysis of ß-adrenergic receptor subtypes in pig tissues. J. Anim. Sci. 80:963–970.[Abstract/Free Full Text]
Liu, C. Y., and S. E. Mills. 1989. Determination of the affinity of ractopamine and clenbuterol for the ß-adrenoceptor of the porcine adipocyte. J. Anim. Sci. 67:2937–2942.
Liu, C. Y., J. L. Boyer, and S. E. Mills. 1989. Acute effects of ß-adrenergic agonists on porcine adipocyte metabolism in vitro. J. Anim. Sci. 67:2930–2936.
McNeel, R. L., and H. J. Mersmann. 1999. Distribution and quantification of ß1-, ß2-, and ß3-adrenergic receptor subtype transcripts in porcine tissues. J. Anim. Sci. 77:611–621.[Abstract/Free Full Text]
Mersmann, H. J., and R. L. McNeel. 1992. Ligand binding to the porcine adipose tissue ß-adrenergic receptor. J. Anim. Sci. 70:787–797.[Abstract]
Mills, S. 2000. ß-Adrenergic receptor subtypes mediating lipolysis in porcine adipocytes. Studies with BRL-37344, a putative ß3-adrenergic agonist. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 126:11–20.
Mills, S. E., and M. E. Spurlock. 1995. Tissue and specie variation in the activation of adenylate cyclase by ß-adrenergic receptors. J. Anim. Sci. 73(Suppl. 1):145. (Abstr.)[Abstract]
Moody, D. E., D. L. Hancock, and D. B. Anderson. 2000. Pheethanolamine repartitioning agents. Pages 65–96 in Farm Animal Metabolism and Nutrition. J. P. F. D’Mello, ed. CAB Int., Wallinford, Oxon, U.K.
Orcutt, A. L., T. R. Cline, and S. E. Mills. 1989. Influence of the ß2-adrenergic agonist clenbuterol on insulin-stimulated lipogenesis in mouse adipocytes. Domest. Anim. Endocrinol. 6:59–69.[Medline]
Ricke, E. A., D. J. Smith, V. J. Feil, G. L. Larsen, and J. S. Caton. 1999. Effects of ractopamine HCl stereoisomers on growth, nitrogen retention, and carcass composition in rats. J. Anim. Sci. 77:701–707.[Abstract/Free Full Text]
Ruffolo, R. R. 1991. Chirality in - and ß-adrenoceptor agonists and antagonists. Tetrahedron 47:9953–9980.
Samama, P., S. Cotecchia, T. Costa, and R. J. Lefkowitz. 1993. A mutation-induced activated state of the ß2-adrenergic receptor: Extending the ternary complex model. J. Biol. Chem. 26:4625–4636.
Shappell, N. W., V. J. Feil, D. J. Smith, G. L. Larsen, and D. C. McFarland. 2000. Response of C2C12 mouse and turkey cells to the ß-adrenergic agonist ractopamine. J. Anim. Sci. 78:699–708.[Abstract/Free Full Text]
Smith, C. K., II, D. E. Lee, and L. L. Coutinho. 1990. Quantitative analysis of the selectivity of ractopamine for ß-adrenergic receptor subtypes. J. Anim. Sci. 68(Suppl. 1):284.
Smith, D. J. 1998. The pharmacokinetics, metabolism, and tissue residues of ß- adrenergic agonists in livestock. J. Anim. Sci. 76:173–194.[Abstract/Free Full Text]
Smith, D. J. 2000. Stereochemical composition of clenbuterol residues in edible tissues of swine. J. Agric. Food. Chem. 48:6036–6043.[Medline]
Smith, D. J., and G. D. Paulson. 1994. Growth characteristics of rats receiving ractopamine hydrochloride and the metabolic disposition of ractopamine hydrochloride after oral or intraperitoneal administration. J. Anim. Sci. 72:404–414.[Abstract]
Spurlock, M. E., J. C. Cusumano, and S. E. Mills. 1993a. The affinity of ractopamine, clenbuterol, and L-644,969 for the ß-adrenergic receptor population in porcine adipose tissue and skeletal muscle membrane. J. Anim. Sci. 71:2061–2065.[Abstract]
Spurlock, M. E., J. C. Cusumano, and S. E. Mills. 1993b. (–)-[3H]-Dihydroalprenolol binding to ß-adrenergic receptors in porcine adipose tissue and skeletal muscle membrane preparations. J. Anim. Sci. 71:1778–1785.[Abstract]
Tate, K. M., M. M. Briend-Sutren, L. J. Emorine, C. Delavier-Klutchko, S. Marullo, and A. D. Strosberg. 1991. Expression of three human ß-adrenergic-receptor subtypes in transfected Chinese hamster ovary cells. Eur. J. Biochem. 196:357–361.[Medline]
Weiland, K., H. M. Zuurmaond, C. Krasel, A. P. Ijzerman, and M. J. Lohse. 1996. Involvement of ASN-293 in stereospecific agonist recognition and in activation of the ß2-adrenergic receptor. Proc. Natl. Acad. Sci. 93:9276–9281.[Abstract/Free Full Text]
Yen, T. T., M. M. McKee, N. B. Stamm, and K. G. Bemis. 1983. Stimulation of cyclic AMP and lipolysis in adipose tissue of normal and obese Avy/a mice by LY79771, a phenethanolamine, and stereoisomers. Life Sci. 32:1515–1522.[Medline]
Top
Abstract
Introduction
