1 The P-adrenoceptor population was characterized in membrane preparations from rat brown adipose tissue (BAT) and from soleus muscle by use of the radioligand ['25I]-iodocyanopindolol (['251I]-ICYP). In addition, atypical binding sites for [12511-ICYP found in both tissues were examined, and the relationship between these sites and the putative rat P3-adrenoceptor is discussed.2 It was established that BAT membranes host a mixed population of l-and P2-adrenoceptors. Of these two sites, 55% showed a high affinity for the P,-selective ligand CGP 20712A (pK 8.5), and 45% showed a high affinity for the p2-selective antagonist ICI 118551 (pK 8.6). Soleus muscle membranes were found to host a population of P2-adrenoceptors, characterized by a high affinity for ICI 118551 (pK 9.1), but ,I-adrenoceptors could not be detected in this preparation. 5-Hydroxytryptamine receptors were not detected in either preparation.3 In addition to Pl-and P2-adrenoceptors, atypical binding sites were identified in both tissues using high concentrations of radioligand (0.5-0.6 nM) and in the presence of 1 tM (-)-propranolol. The atypical sites were abundant, representing 80 and 81% of the total ['25I]-ICYP binding sites in BAT and soleus muscle respectively. When the pK values for 11 ligands were compared, the correlation coefficient for atypical sites in BAT and soleus muscle was 0.94. 4 The atypical binding sites showed a moderate affinity for (±)-cyanopindolol (pK 7.3-7.7), poor stereoselectivity for the (+)-and (-)-enantiomers of alprenolol (<10 fold), and a low affinity for B-adrenoceptor antagonists and partial agonists in the order: (±)-cyanopindolol>(-)-alprenolol> (-)-propranolol=(±)-ICI 118551>>(±)-CGP20712A. The affinity of these ligands for the atypical sites reflects their behaviour in functional studies of putative P3-adrenoceptors in rat BAT, white adipose tissue, intestine and colon.5 The atypical sites labelled by [1251]-ICYP were resistant to agonist binding, and while the order of affinity of the agonists BRL 37344> isoprenaline> noradrenaline matches their order of potency at putative P3-adrenoceptors, none of these compounds caused displacement of the radioligand at concentrations below 10 DIM. 6 It is concluded that the atypical binding sites for ['251]-ICYP found in rat BAT and soleus muscle membranes are the same, and that these sites show some relationship to the putative rat P3-adrenoceptor identified in functional studies using antagonists. However, under the conditions used in the present study, pK values obtained for 33-agonist binding are not useful.

It is reported that a long duration of action is required for beta 2-adrenoceptor agonists to evoke an anabolic response. In the present study, we compare the potency of clenbuterol with that of the new long-acting compound salmeterol, when given at equimolar doses to female Wistar rats by different routes of administration. Given orally for 10 days, salmeterol had no effect on growth at a dose of 120 micrograms/day, whereas at 2.4 mg/day the drug caused significant increases in body and carcass weight and in the mass of the mixed-fiber gastrocnemius/plantaris and tibialis anterior muscles, but there were no increases in the slow-twitch soleus muscles. A similar growth response was seen when clenbuterol was given orally at a dose of only 97 micrograms/day, with an additional response seen in soleus muscle at 1.9 mg/day. Thus clenbuterol was more potent than salmeterol when given by this route of administration. When the drugs were infused by osmotic minipump, both salmeterol (130 micrograms/day) and clenbuterol (100 micrograms/day) caused increases in body weight gain and in the weights of the mixed-fiber muscles, with the most dramatic effect of infusion being to greatly increase the anabolic effect of salmeterol in soleus muscle. A single intraperitoneal injection of salmeterol (53 micrograms) or clenbuterol (40 micrograms) caused a similar rapid increase in the concentration of adenosine 3',5'-cyclic monophosphate in gastrocnemius muscle. These results indicate that the potency of salmeterol in vivo is dependent on its route of administration and that slow-twitch muscles are less sensitive than mixed-fiber muscles to the anabolic effects of beta 2-adrenoceptor agonists.

The morpholine compound BRL-47672 has a chemical structure similar to that of clenbuterol and causes similar anabolic effects in rats but has no actions on beta 2-adrenoceptors in vitro. It has been argued therefore that beta 2-adrenoceptors do not mediate the anabolic effects of this family of compounds. In the present study BRL-47672 was shown to bind to rat beta 2-adrenoceptors with low affinity (dissociation constant 16 microM) relative to clenbuterol (48 nM) and to be a very weak activator of adenylyl cyclase activity in rat skeletal muscle membranes in vitro. In contrast, acute administration of the drug to anesthetized rats in vivo caused an increase in muscle adenosine 3',5'-cyclic monophosphate output, and chronic treatment of conscious rats for > 6 days caused a significant increase in weight gain (69%) accounted for by increased muscle growth. The anabolic effects of BRL-47672 were not counteracted by daily injections of the drug ICI-118551 (2 mg/day) but were prevented when the same beta 2-antagonist was administered in the diet (200 mg/kg feed, equivalent to 4.3 mg/day). The beta 1-adrenoceptor selective antagonist CGP-20712A fed in the diet (200 mg/kg feed) failed to attenuate the response to BRL-47672. These results support the conclusion that BRL-47672 has little direct action on beta 2-adrenoceptors but suggest that the compound is metabolized rapidly in vivo to a potent beta 2-agonist. Thus the stimulation of muscle growth by BRL-47672 is via beta 2-adrenoceptors, with no contribution to this response from beta 1- or beta 3-adrenoceptor activation.

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