Figure 3.

Distribution of Muscarinic Receptors. (A) Immunohistochemical localisation of endothelial M3 muscarinic acetylcholine receptors (AChRM3s) in the endothelium. Representative image (top left) illustrating that the AChRM3 distribution was not uniform across the endothelium but was more densely clustered in discrete regions (top right, yellow lines). In the same preparation, the nuclei of endothelial cells were labelled with DAPI (bottom left). An overlay (bottom right) of endothelial nuclei (blue) with AChRM3 (red) staining shows the clustered localisation of AChRM3s in particular regions of the endothelium (bottom right, yellow lines). (B) Negative control obtained by omitting anti-AChRM3 (top). DAPI loading (bottom) shows the positions of cell nuclei. (C) AChRM3s are increased in the most-sensitive cells activated by acetylcholine (ACh). Left: Total endothelial Ca²⁺ activity (green; evoked by 30-nM ACh) overlaid on cells (grey) in a carotid artery preparation. Right: Immunohistochemical localisation of endothelial AChRM3s (red) in the same field of endothelium from which the Ca²⁺ signals were obtained. Nuclei are shown in blue (DAPI staining). (D) Summary data showing that AChRM3s are more densely localised in ACh-sensitive regions of endothelium [i.e., green in (C)] compared with regions that are less sensitive (n = 3, P < 0.05). All bars, 50 μm. Reproduced from [42].