Functional compartmentation of endothelial P2Y receptor signaling

The presence of multiple receptors for disparate nucleotides on endothelial cells makes it unclear how the endothelium differentiates among these signals. We propose that endothelial P2Y receptors are organized into cholesterol-rich signaling domains, such as caveolae and respond to nucleotide agonists by mobilizing intracellular calcium. Treatment of endothelial cells with 5 mmol/L beta-methyl-cyclodextrin prevents calcium release in response to the nucleotide receptor agonists 2-methylthio-ATP, ATP, ADP, and UTP, but not the kinin receptor agonist bradykinin, suggesting that depletion of membrane cholesterol disrupts signaling at P2Y receptors and that bradykinin receptors are not prelocalized to cholesterol microdomains in these cells. Direct measurement of cholesterol content after beta-methyl-cyclodextrin treatment of aortic rings reveals a concentration-dependent depletion of cholesterol that parallels functional antagonism of P2Y-mediated relaxation. Nucleotide- and bradykinin-mediated relaxation is disrupted by 5 to 15 mmol/L beta-methyl-cyclodextrin treatment or 1 to 10 mug/mL filipin III in a concentration-dependent fashion. Norepinephrine contracted aorta treated with A23187 relaxes in an endothelium-dependent fashion despite depletion of 84% of membrane-extractable cholesterol. These data indicate that in the basal state, P2Y receptors but not the kinin receptor may be compartmented to cholesterol-dependent signaling domains in guinea pig endothelium and that cholesterol-rich microdomains in these cells can respond to intracellular calcium in an agonist-specific manner. We suggest that the functional organization of cholesterol-rich signaling microdomains allows agonist-specific responses to increases in intracellular calcium and that this property may be a general phenomenon that permits cells to respond disparately to agonists that may signal through common calcium release pathways.