Evaluation of β1L-Adrenoceptors in Rabbit Heart by Tissue Segment Binding Assay

Abstract

Abstract. [3 H]-CGP12177 biphasically bound to β-adrenoceptors with high and low affinities in the segments and crude membranes of rabbit left ventricle. The low-affinity sites for [ 3 H]-CGP12177 in the segments was double in density, compared to the density of high-affinity sites. Total abundance of the β-adrenoceptors decreased to approximately 10% upon tissue homogenization, and the proportion of low-affinity sites was the same as that of the high-affinity sites in the membranes. The majority of the high-affinity binding sites of [ 3 H]-CGP12177 in the segments and the membranes were β 1H -adrenoceptor, being highly sensitive to propranolol and β 1 -antagonists (atenolol and ICI-89,406), whereas the low-affinity binding sites showed a β 1L -profile (less sensitive to propranolol and β 1 -, β 2 -, and β 3 -antagonists). Furthermore, a part of the β 1L -adrenoceptors was insensitive to atenolol, ICI-89,406, and/or isoproterenol. The present binding study clearly shows that β 1L -adrenoceptors occur as a distinct phenotype from β 1H -adrenoceptors in rabbit ventricle. However, quantitative imbalance between β 1H -and β 1L -adrenoceptors and divergent ligand-β 1L -adrenoceptor interactions suggest a possibility that the β 1L -adrenoceptor may not reflect a simple conformational change or allosteric state in the β 1 -adrenoceptor molecule.

Influence of Tissue Integrity on Pharmacological Phenotypes of Muscarinic Acetylcholine Receptors in the Rat Cerebral Cortex

“…Binding data were analyzed using Prism (version 5.01; GraphPad Software Inc., San Diego, CA) as described previously (Muramatsu et al, 2005;Yoshiki et al, 2009). In brief, the data from saturation binding studies were fitted by a one-site saturation binding isotherm (binding-saturation equation in Prism), and the K d values and the binding capacity were then calculated.…”

Assessment of Novel Muscarinic Acetylcholine Receptors in Rat Cerebral Cortex by a Tissue Segment Binding Method

“…The cerebral cortex was cleaned from the pia mater and substantia alba and cut into small pieces (approximately 2 × 2 × 1 mm). Tissue segment binding experiments were performed at 37°C or at 4°C, according to the previously described method (7,16 …”

Re-Evaluation of Nicotinic Acetylcholine Receptors in Rat Brain by a Tissue-Segment Binding Assay

“…Binding data were analyzed using PRISM software (Version 5.01, Graph Pad Software, La Jolla, CA, USA), as previously described (Muramatsu et al, 2005; Yoshiki et al, 2009). Briefly, the data from saturation binding studies were fitted by a one-site saturation binding isotherm and the K d values and the binding capacity were then calculated.…”

“…These recent studies suggest that the in situ environment significantly contributes to some receptor properties. To avoid artificial modification of the receptor environment as much as possible, a binding assay for use with intact tissue segments and with physiological solution (such as Krebs solution) was recently developed and has been demonstrated to be a powerful method for detecting the inherent profiles of receptors in a native manner (Tanaka et al, 2004; Muramatsu et al, 2005; Yoshiki et al, 2009; Anisuzzaman et al, 2011). In contrast to the conventional membrane binding assay, the tissue-segment binding assay is less prone to have a yield loss of receptors and to have a modification of the natural environment/conformation of receptors, which may be caused during tissue homogenization and membrane fractionation.…”