Fig. 8. Heterogenous distribution of muscarinic and purinergic receptors.

(A) Fluorescence localization of purinergic P2Y2 receptors and muscarinic M3 receptors in the endothelium. Representative images (from left) show the endothelial cell boundaries as revealed by PECAM-1 labelling (green; anti- CD31/PECAM-1), P2Y2 receptor (red; anti-P2Y2) distribution, M3 receptor distribution (green; fluorescent M3 receptor antagonist, 100 nM) and overlay of all three. The receptors distribution was not uniform across the endothelium and there was relatively little overlap of purinergic and muscarinic receptor staining. (B) Expanded view of the endothelial images shown in A. The expanded region is shown by the red box in A left-panel. (C) Negative control. The left panel is again PECAM-1 labeled to show cell boundary positions. The negative control for P2Y2 staining was obtained by omitting the primary antibody against P2Y2. The control for M3 staining was obtained by labelling with the fluorescent M3 receptor ligand in the presence of the M3 receptor antagonist 4-DAMP (10 μM). The right panel shows an overlay of all three. (D) Summary data showing the levels of M3 staining in the presence and absence of the antagonist 4-DAMP (green), the extent of labelling in the presence and absence of the P2Y2 primary antibody (red); and the extent of overlap of specific M3 and P2Y2 staining (grey). (E) Summary data showing the percentage of cells with M3 staining (green), P2Y2 receptor staining (red) and the percentage of cells expressing both M3 and P2Y2 receptor staining (grey) (n = 3, *p < 0.05, unpaired t-test). Scale bars, 50 μm.