Fig 1.
Study design of (A) competition radioligand binding assay to quantify β adrenergic receptor (AR) subtypes in whole lung of (B) wild-type C57BL/6J (WT), (C) β-arrestin-1 knockout (βarr1 KO), and (D) β-arrestin-2 knockout (βarr2 KO) mice.
A. Competitive displacement of a non-selective antagonist radioligand from a mixed population of receptors (50:50) by a subtype-selective competitor was simulated. Data were generated by fitting affinities of the antagonist ICI-118551 for the β2AR (Log Kd = -9.26) and the β1AR (Log Kd = -6.52) [23] to a two-site competitive binding model in GraphPad Prism. Due to its >500-fold selectivity for the β2AR, ICI-118551 displaces radioligand from β2ARs at low concentrations and from β1ARs at high concentrations to produce a biphasic inhibition curve. The deconvolution of high and low affinity states quantifies the fraction of each receptor subtype. In the case of ICI-118551, subtype 1 represents the β2AR and subtype 2 represents the β1AR. B-D. Competition binding between (125I)-CYP (60 pM) and ICI-118551 (0.3 pM to 10 μM) detected 36% β1AR and 64% β2AR in WT mouse whole lung (B), 43% β1AR and 57% β2AR in βarr1 KO whole lung (C), and 33% β1AR and 67% β2AR in βarr2 KO whole lung (D). Binding parameters can be found in Table 2, with the data representing the mean ± SEM of 3–6 independent experiments performed in duplicate.
Fig 2.
Quantification of β adrenergic receptor (AR) subtypes from a mixed population of βARs using calibrated concentrations of the β1AR-selective antagonist CGP-20712A and the β2AR-selective antagonist ICI-118551.
A. Proof-of-concept saturation experiments with β1AR-overexpressing membranes demonstrate that 500 nM CGP-20712A completely displaces (125I)-CYP from all available β1ARs, whereas 100 nM ICI-11855 is sufficiently low to not detect the β1AR. Total β1AR was set to 100% based on the displacement of (125I)-CYP by 10 μM propranolol. B. Proof-of-concept saturation experiments with β2AR-overexpressing membranes demonstrate that 100 nM ICI-118551 completely displaces (125I)-CYP from all available β2ARs, whereas 500 nM CGP-20712A is sufficiently low to not detect the β2AR. Total β2AR was set to 100% based on the displacement of (125I)-CYP by 10 μM propranolol. Plotted data represent the individual means of three experiments performed in duplicate. Data were fit to a one-site saturation model in GraphPad Prism.
Table 1.
β adrenergic receptor (AR) subtype levels (%) in murine lung and epithelia-denuded tracheobronchial smooth muscle.
Table 2.
The affinities of ICI-118551 at β2 adrenergic receptor (AR) (pKHi) and β1AR (pKLo) in murine lung and expression values (fmol/mg) of βAR subtypes in lung and epithelia-denuded tracheobronchial smooth muscle.
Fig 3.
Estimation of β adrenergic receptor (AR) subtypes by single-point saturation binding assay in whole lung of (A) wild-type (WT) C57BL/6J, (B) β-arrestin-1 knockout (βarr1 KO), and (C) β-arrestin-2 knockout (βarr2 KO) mice.
The competitive displacement of the non-selective radiolabeled antagonist (125I)-cyanopindalol (CYP) (500 pM) by 500 nM CGP-20712A and 100 nM ICI-118551 quantifies the proportions of β1AR and β2AR, respectively. Propranolol, a nonselective βAR blocker, gives a measure of total βAR present in each tissue. A. WT: β1AR = 32 ± 3%; β2AR = 67 ± 2%; 100% corresponds to 887.2 ± 168 fmol/mg. B. βarr1 KO: β1AR = 38 ± 1%; β2AR = 63 ± 2%; 100% corresponds to 1072 ± 222 fmol/mg. C. βarr2 KO: β1AR = 32 ± 0.5%; β2AR = 61 ± 2%; 100% corresponds to 1221 ± 277 fmol/mg. Data represent the mean ± SEM of 3 independent experiments performed in quadruplicate.
Fig 4.
Estimation of β adrenergic receptor (AR) subtypes by single-point saturation binding assay in tracheobronchial smooth muscle of (A) wild-type (WT) C57BL/6J, (B) β-arrestin-1 knockout (βarr1 KO), and (C) β-arrestin-2 knockout (βarr2 KO) mice.
The competitive displacement of the non-selective radiolabeled antagonist (125I)-cyanopindalol (CYP) (500 pM) by 500 nM CGP-20712A and 100 nM ICI-118551 quantifies the proportions of β1AR and β2AR, respectively. Propranolol, a nonselective βAR blocker, gives a measure of total βAR present in each tissue. A. WT: β1AR = 12 ± 5%; β2AR = 64 ± 3%; 100% corresponds to 208.2 ± 28 fmol/mg. B. βarr1 KO: β1AR = 13 ± 4%; β2AR = 60 ± 4%; 100% corresponds to 213 ± 55 fmol/mg. C. βarr2 KO: β1AR = 14 ± 4%; β2AR = 65 ± 2%; 100% corresponds to 255.7 ± 82 fmol/mg. Data represent the mean ± SEM of 3 independent experiments performed in quadruplicate.