Assessment of Muscarinic Receptor Subtypes in Human and Rat Lower Urinary Tract by Tissue Segment Binding Assay

Abstract

Abstract. Muscarinic receptors in the human and rat lower urinary tract (urinary bladder detrusor muscle and mucosa, and prostate) were identified by intact tissue segment binding assays with two radioligands, and the effects of prolonged receptor activation in vitro on muscarinic receptors were examined. Hydrophilic [3 H]-NMS and hydrophobic [ 3 H]-QNB bound to the detrusor muscle segments with the same density, suggesting that the muscarinic receptors were localized at the plasma membrane. While the density of muscarinic receptor was higher in detrusor muscle than in the bladder mucosa and prostate, there was no species-specific difference either in density or in subtype distribution (M 1 , M 2 , and M 3 subtypes in detrusor; M 2 and M 3 subtypes in bladder mucosa; and M 1 and M 2 subtypes in prostate). Incubation of detrusor strips with carbachol decreased [3 H]-NMS binding sites within 20 min, followed by a reduction of [ 3 H]-QNB binding sites after a 60-min lag phase. The loss of the binding sites over 3 h after carbachol treatment was the same (approximately 40%) for both radioligands. The present intact tissue segment binding assay reveals tissue-specific and plasma membrane distribution of distinct muscarinic receptor subtypes and their dynamic changes (internalization and down-regulation) in lower urinary tract of humans and rats.

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

“…The tissues were cut into small pieces (approximately 2 ϫ 2 ϫ 1 mm for cerebral cortex, hippocampus, and corpus striatum, approximately 1.5 ϫ 1.5 ϫ 1 mm for gastric muscle and bladder muscle, and approximately 4 ϫ 3 ϫ 3 mm for midbrain, pons, and submaxillary gland) and applied to tissue segment binding experiments. The binding incubation for tissue segments was routinely performed at 4°C for 16 to 18 h in a final volume of 1 ml (Muramatsu et al, 2005;Anisuzzaman et al, 2008b). To ensure equilibrium in binding, we adopted the fail-safe assumption, and experiments of longer incubation (32 or 48 h) were also performed (see Results and Supplemental Figs.…”

Contractions of the Mouse Prostate Elicited by Acetylcholine Are Mediated by M3 Muscarinic Receptors

“…Both the M 1 and M 2 muscarinic receptor subtypes have been observed in binding studies of whole human prostate homogenates (Anisuzzaman et al, 2008). However, Ruggieri et al (1995) observed only the M 1 muscarinic subtype, which was found to be localized immunohistochemically in the epithelium.…”

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

“…[ 3 H]-CGP12177 was previously validated as a radioligand to label the β 1H -and β 1L -adrenoceptors, in addition to β 2 -and β 3 -adrenoceptors (18). Since the tissue segment binding assay has been recently demonstrated to be a powerful method to detect native receptors without changing receptor environment (16,17), the present study was designed to identify the native states of β 1H -and β 1L -adrenoceptors in the rabbit ventricle, and the native profile in the segments was compared with the profile obtained in the conventional membrane binding assay. The saturation binding isotherm with [ H]-CGP12177 at both sites were not different between the segments and the membranes, but the total density (B max value) of β-adrenoceptors was significantly lower (approximately 10%) in the membrane preparations than in the segments (Fig.…”

“…4). This represents a large yield loss of receptor by tissue homogenization, as reported in other tissues (16,17,20,21). Another difference between the tissue segment binding assay and the conventional membrane binding assay was the density ratio of high-and low-affinity sites for […”

“…Recently, we have developed an intact tissue segment binding method to detect native receptors (16,17). This approach without homogenization is an extremely powerful tool for detecting the native states of receptors with less modification.…”