Stereoselectivity of porcine β-adrenergic receptors for ractopamine stereoisomers^,

ABSTRACT

Ractopamine HCl is a β-adrenergic receptor (βAR) 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 βAR subtype selectivity, receptor affinity and adenylyl cyclase activation were determined using cloned porcine β₁- and β₂AR expressed in Chinese hamster ovary (CHO) cells. Dissociation constants (K_d) were determined by competitive displacement of [¹²⁵I]iodocyanopindolol binding by ractopamine stereoisomers. The RR isomer had the highest affinity for both β₁- and β₂AR (K_d 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 β₁- and β₂AR, respectively) relative to the RR stereoisomer. Isoproterenol stimulated adenylyl cyclase activity 600% relative to basal rates in CHO cells, regardless of βAR subtype. Ractopamine stereoisomers did not significantly (P > 0.05) stimulate adenylyl cyclase through the β₁AR at moderate (near K_d) or high (10⁻⁴M) concentrations. In contrast, the RR isomer increased adenylyl cyclase activity 200 to 300% relative to basal rates through the β₂AR 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 β₂AR. 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 βAR exhibited stereoselectivity toward ractopamine stereoisomers with the RR isomer exhibiting the highest affinity for the β₁- and β₂AR. In contrast, ractopamine stereoisomers seemed to be more effective at eliciting adenosine cyclic 3′,5′-phosphate responses from β₂AR than β₁AR. 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.

β-Adrenergic Receptor, Growth, Pigs