[3H]-L 748,337, a novel radioligand for the labelling of β3-adrenoceptors

Jan-Peter van Wieringen, Martina B Michel-Reher, Martin C Michel. AMC, Dept. Pharmacology & Pharmacotherapy, 1105AZ Amsterdam, Netherlands.

β3-Adrenoceptors are expressed in several tissues at the mRNA level but little is known about their presence at the protein level. The reason is that until now functional studies often have remained ambiguous due to lack of selectivity of the employed pharmacological tools, whereas direct protein detection studies were hampered by lack of selectivity of antibodies and the absence of a radioligand which labels this subtype with similar or higher affinity than β1- and β2-adrenoceptors. L 748,337 is an antagonist with selectivity for β3- over β1- and β2-adrenoceptors (Ki 4 vs. 390 and 204 nM, respectively; Candelore et al., 1999). Therefore, we have explored whether a radiolabelled version L 748,337 may be a suitable radioligand to label β3-adrenoceptors.

L 748,337 ((S)-N-[4-[2-[[3-[3-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]-ethyl]phenyl]-benzenesulfonamide) was custom tritiated by Perkin Elmer (Zaventem, Belgium) to a specific activity of 34 Ci mmol-1. Saturation and competition binding experiments were performed with membranes prepared from Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells stably transfected with human β-adrenoceptor subtypes. Equilibrium binding experiments were performed for 60 min at 25°C in binding buffer (10 mM Tris, 154 mM NaCl at pH 7.4) in 96 well polystyrene plates. Non-specific binding was defined by 100 µM isoprenaline, and incubations were terminated by rapid vacuum filtration on GF/C glass fibre filter plates. Saturation and competition binding data were fitted to hyperbolic and sigmoidal functions, respectively.

In HEK and CHO cells expressing human β3-adrenoceptors, [3H]-L 748,337 exhibited saturable specific binding with a Kd of 3.4 ± 1.2 and 3.7 ± 0.7 nM and a Bmax of 360 ± 58 and 197 ± 15 fmol mg protein-1 (n = 5-6). In β3-adrenoceptor-expressing HEK cells, noradrenaline, adrenaline, isoprenaline, CGP 20,712A (2-hydroxy-5-(2-(hydroxy-3-(4((1-methyl-4-trifluoromethyl)-1-H-imidazol-2-yl)-phenoxy)-propyl)-aminoethoxyl)-benzamide), ICI 118,551 (3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol) and non-tritiated L 748,337 competed for radioligand binding with a pattern compatible with the labelling of β3-adrenoceptors.

As non-specific binding had been unsatisfactorily high in the above experiments, we performed parallel saturation binding experiments in HEK cells with GF/C and GF/B filters, each in the absence and presence of pre-coating with 0.1% polyethylenimine. While GF/B filters did not exhibit less non-specific binding than GF/C filters (GF/C 39 ± 3%, GF/B 50 ± 3%; n = 4) polyethylenimine coating markedly reduced non-specific binding with both filter types (GF/C 20 ± 0.4%, GF/B 27 ± 2%) without major effects on estimated Kd (2.7 ± 0.8, 2.7 ± 0.4, 2.1 ± 0.2 and 2.0 ± 0.3 nM, respectively) and Bmax values (551 ± 29, 623 ± 55, 718 ± 61 and 644 ± 60 fmol mg protein-1, respectively).

Under these conditions [3H]-L 748,337 in concentrations up to 20 nM did not yield saturable binding to membranes from CHO cells transfected with β1- or β2-adrenoceptors; while some specific binding was observed, it was considerably less than we previously found to be expressed in these cells (Niclauss et al., 2006).

We conclude that [3H]-L 748,337 may be suitable for the labelling of human β3-adrenoceptors, specifically if used in conjunction with polyethylenimine-coated GF/C filters.

Candelore, MR et al. (1999) J Pharmacol Exp Ther 290: 649-655

Niclauss, N et al. (2006) Naunyn-Schmiedeberg’s Arch Pharmacol 374: 79-85.

This study was funded in part by Astellas Pharma B.V. (Leiderdorp, The Netherlands).