Graham Pegg

Ligand binding properties of putative β3‐adrenoceptors compared in brown adipose tissue and in skeletal muscle membranes

1 The β‐adrenoceptor population was characterized in membrane preparations from rat brown adipose tissue (BAT) and from soleus muscle by use of the radioligand [125I]‐iodocyanopindolol ([125I]‐ICYP). In addition, atypical binding sites for [125I]‐ICYP found in both tissues were examined, and the relationship between these sites and the putative rat β3‐adrenoceptor is discussed. 2 It was established that BAT membranes host a mixed population of β1‐ and β2‐adrenoceptors. Of these two sites, 55% showed a high affinity for the β1‐selective ligand CGP 20712A (pK 8.5), and 45% showed a high affinity for the β2‐selective antagonist ICI 118551 (pK 8.6). Soleus muscle membranes were found to host a population of β2‐adrenoceptors, characterized by a high affinity for ICI 118551 (pK 9.1), but β1‐adrenoceptors could not be detected in this preparation. 5‐Hydroxytryptamine receptors were not detected in either preparation. 3 In addition to β1‐ and β2‐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 μm (−)‐propranolol. The atypical sites were abundant, representing 80 and 81% of the total [125I]‐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 β‐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 β3‐adrenoceptors in rat BAT, white adipose tissue, intestine and colon. 5 The atypical sites labelled by [125I]‐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 β3‐adrenoceptors, none of these compounds caused displacement of the radioligand at concentrations below 10 μm. 6 It is concluded that the atypical binding sites for [125I]‐ICYP found in rat BAT and soleus muscle membranes are the same, and that these sites show some relationship to the putative rat β3‐adrenoceptor identified in functional studies using antagonists. However, under the conditions used in the present study, pK values obtained for β3‐agonist binding are not useful.

Characteristics of cyanopindolol analogues active at the β3‐adrenoceptor in rat ileum

1 Cyanopindolol (CYP) is a potent antagonist at the β3‐adrenoceptor in rat ileum. Several analogues of CYP and pindolol were synthesized that also produced antagonist effects at the β3‐adrenoceptor. However, at high concentrations, these compounds appear to act as ‘partial agonists’. This study was conducted to determine the structural requirements of CYP analogues necessary for antagonist activity and to examine the possibility that the agonist effects of CYP and its analogues may occur through a mechanism independent of β‐adrenoceptor activation. 2 Analogues of CYP and pindolol were tested for antagonist activity in rat ileum in which the β1‐ and β2‐adrenoceptors were blocked. Fourteen compounds were tested against (−)−isoprenaline, and four of the more potent analogues were then tested against BRL 37344. The two most potent antagonists were CYP and iodocyanopindolol. The pKb values (negative log of equilibrium dissociation constant) obtained against (−)−isoprenaline were significantly higher than those obtained against BRL 37344, but the cause of this difference is not known. 3 Several structural requirements were determined for antagonist activity. Modification at the carbon atom alpha to the secondary amine caused the antagonist potency to fall as the level of saturation was reduced. Thus, a quaternary carbon group, such as t‐butyl, produced the most potent antagonist. Substitution with a large moiety such as a cyclohexyl or benzyl group reduced antagonist activity, probably due to steric hindrance. Inclusion of an electron‐withdrawing group, such as a cyano or ethylester moiety, alpha to the indole nitrogen, also increased the potency. Iodination of CYP and ethylesterpindolol at the 3‐position of the indole ring did not increase antagonist potency. In contrast, iodination of the almost inactive analogues produced a significant increase in potency, suggesting that a beneficial electronic effect on the indole ring imparted by the iodo moiety may be able to offset partially the negative effects caused by either the steric hindrance, of lack of a quaternary carbon alpha to the secondary amine. 4 Values for pseudo‐pD2 were also determined by conducting cumulative concentration‐response studies up to the limit of drug solubility. For nine of the compounds tested, the pKb was significantly higher than the pseudo‐pD2 value. 5 The discrepancy between the pKb and pseudo‐pD2 values was examined further. The agonist effects of iodocyanopindolol, the agonist with the highest potency, were not antagonized by CYP which was the most potent antagonist of (−)−isoprenaline and BRL 37344 at the β3‐adrenoceptor. This suggests that the agonist effects of iodoCYP were produced through a different mechanism: either via another receptor, another isoform of the rat β3‐adrenoceptor, or through a non‐receptor‐mediated effect. Pseudo‐pD2 values did not correlate with log P values for these compounds, indicating that their relaxant effects were not simply a function of their lipid solubility. 6 This study has highlighted several structural requirements for antagonist binding potency at the rat ileum β3‐adrenoceptor and should assist in the development of potent selective antagonists for this receptor.

Atypical responses of rat ileum to pindolol, cyanopindolol and iodocyanopindolol.

1 Pindolol, cyanopindolol (CYP) and iodocyanopindolol (IodoCYP) have been reported to act either as antagonists, agonists or partial agonists at the β3‐adrenoceptor in different preparations. A comprehensive investigation has not yet been described with these compounds tested in one tissue from one species. This study was conducted to delineate the pharmacological effects of pindolol, CYP and IodoCYP and to provide data on their affinities at the predominant β‐adrenoceptor in rat ileum. 2 The β‐adrenoceptors present in rat ileum were characterized in the presence of CGP 20712A and ICI 118 551, atropine and corticosterone, with (−)−isoprenaline used as an agonist. The role of the β1 and β2‐adrenoceptors was determined by the omission of either CGP 20712A, ICI 118 551, or both, from the buffers. Conversely, the effectiveness of the β1‐ and β2‐adrenoceptor blockade was examined by use of the β1‐adrenoceptor‐selective agonist, RO 363 and the β2‐adrenoceptor‐selective agonist, salbutamol. 3 There was no evidence for the presence of functional β1‐adrenoceptors, and no strong evidence that β2‐adrenoceptor stimulation contributed to the relaxant effects of (−)−isoprenaline. (−)−Phenylephrine did not produce relaxation of the tissue and 5‐hydroxytryptamine produced contraction. 4 The β3‐adrenoceptor‐selective agonist, BRL 37344 and (−)−isoprenaline were potent full agonists (pD2 8.35 ± 0.04 and 7.76 ± 0.14 respectively), whereas ICI D7114 was less potent (pseudo pD2 6.92 ± 0.15). These results indicate that the predominant functional β‐adrenoceptors in rat ileum are β3‐adrenoceptors. 5 Partial agonist effects were produced by CYP (pD2 5.28 ± 0.26) and IodoCYP (pD2 7.0 ± 0.26), but not pindolol. All three compounds antagonized the effects of (−)−isoprenaline with pKb values of 6.68 ± 0.10, 7.59 ± 0.07 and 7.59 ± 0.11 for pindolol, CYP and IodoCYP respectively. Likewise, CYP and IodoCYP antagonized the effects of BRL 37344 with pKb values of 7.20 ± 0.22 and 7.21 ± 0.14 respectively. This study provides the first functional data on the effects of IodoCYP, the ligand with the highest known affinity for the β3‐adrenoceptor, at the characterized rat ileum β3‐adrenoceptor. 6 In conclusion, whereas pKb values suggest that CYP and IodoCYP have a similar affinity for the β3‐adrenoceptor in rat ileum, the higher potency of IodoCYP suggests that it promotes a greater coupling efficiency, or that its partial agonist effects are produced through a site other than the β3‐adrenoceptor. The similar pKb values for CYP and IodoCYP at the β3‐adrenoceptor contrast with their order of known affinities at the β1 and β2‐adrenoceptors, where IodoCYP is far more potent than CYP. This provides evidence of further differences in the characteristics of the β3‐adrenoceptors compared to the β1‐ and β2‐adrenoceptors. Finally, the utility of IodoCYP as a β3‐adrenoceptor antagonist would appear to be limited because of the greater magnitude of partial agonist effects that it produces.