Can [125I]-Iodocyanopindolol Label β3-Adrenoceptors in Rat Urinary Bladder?

Abstract

β3-Adrenoceptors have been demonstrated to mediate urinary bladder smooth muscle relaxation but proof of their expression at the protein level has been missing because of lack of suitable antibodies or radioligands. As among various available radioligands [125I]-iodocyanopindolol ([125I]-ICYP) exhibited the smallest problems in labeling cloned human β3-adrenoceptors in previous studies, we have explored its suitability to label β3-adrenoceptors in rat urinary bladder in saturation and competition radioligand binding experiments. Rat lung was used as an internal control and exhibited all characteristics expected from this tissue with regard to β1/β2-adrenoceptor labeling. Saturation and competition binding studies with [125I]-ICYP in rat bladder yielded saturable binding sites with an affinity compatible with β3-adrenoceptors. In competition experiments various agonists and antagonists largely exhibited a profile compatible with a population consisting largely of β3-adrenoceptors. However, the binding competition properties of ICI 118,551 and SR 59,230A were not easily explained by the idea of labeling a homogeneous β3-adrenoceptor population but interpretation of the data was limited by a high degree of non-specific binding in [125I]-ICYP concentrations required to label the receptors. We conclude that [125I]-ICYP can be used to label tissue β3-adrenoceptors but results obtained with this ligand have to be interpreted with caution.

Rat β3-adrenoceptor protein expression: antibody validation and distribution in rat gastrointestinal and urogenital tissues

“…only 1/1552 of values in BAT, in line with previous reports that rat lung lacks β 3 -adrenoceptor mRNA (Muzzin et al 1991) or protein expression (Schneider and Michel 2010). Accordingly, Sc1473 did not yield detectable staining in rat lung in immunohistochemical experiments but distinct staining in transitional epithelium and inner longitudinal muscularis of rat bladder (data not shown).…”

“…Thus, β 3 -adrenoceptors have not been detected in rat (Muzzin et al 1991) or mouse lung (Nahmias et al 1991) at the mRNA level and in rats based on radioligand binding studies at the protein level (Schneider and Michel 2010); lack of β 3 -adrenoceptor mRNA expression in rat lung was confirmed in the present study. Accordingly, Sc1473 stained neither rat nor murine lung but, in parallel experiments, exhibited distinct staining in mouse heart or rat urinary bladder, which are known to express corresponding mRNA.…”

Agonist‐induced desensitisation of β3‐adrenoceptors: Where, when, and how?

“…In transfected cells, not expressing any of the other subtypes, [ 125 I]‐iodocyanopindolol appears more suitable and is routinely used (Emorine et al, ; Hoffmann et al, ; Niclauß et al, ; Vrydag et al, ). It has also been used in some tissues with native expression of the receptor, but the considerable non‐specific binding has limited interpretation of the data (Hutchinson, Chernogubova, Sato, Summers, & Bengtsson, ; Rathi et al, ; Roberts, Molenaar, & Summers, ; Rouget et al, ; Schneider & Michel, ). [ 3 H]‐SB 206,606, a tritiated form of the active RR‐enantiomer of the agonist BRL 37,344, has been introduced as a β 3 ‐adrenoceptor‐selective radioligand (Muzzin et al, ) but the reported K d values of 38–93 nM limit its use.…”

The pharmacological rationale for combining muscarinic receptor antagonists and β-adrenoceptor agonists in the treatment of airway and bladder disease

“…Moreover, the relative contribution of β-adrenoceptor subtypes at the mRNA level may be different in human urothelium [ 66 ]. The relative contribution of subtypes to total bladder β-adrenoceptor expression at the protein level has been more difficult to determine due to a lack of suitable radioligands or antibodies [ 72 ], but some radioligand binding studies have suggested that mostly β 3 -adrenoceptors may be present [ 73 ]. On the basis of more recently emerging antibody validation data [ 74 • ], the presence of β 3 -adrenoceptors in the human bladder has also been demonstrated by immunohistochemistry, surprisingly showing an apparently greater abundance in urothelium than in smooth muscle [ 75,76 ].…”