Selective measurement of endothelial or smooth muscle [Ca 2+] i in pressurized/perfused cerebral arteries with fura-2

Abstract

Despite a critical role for calcium in endothelial regulation of cerebrovascular tone, endothelial intracellular calcium ([Ca 2+] i) has never been measured in the context of an intact pressurized cerebral vessel. The purpose of the present study was to selectively measure endothelial or smooth muscle [Ca 2+] i and diameter in a pressurized/perfused cerebral vessel. In a pressurized rat middle cerebral artery, fura-2 AM was administered selectively to either the luminal (endothelium) or abluminal (smooth muscle) side of the vessel. Selectivity of loading was determined by measuring fura-2 fluorescence before and after removal of the endothelium. Removal of the endothelium virtually eliminated fura-2 fluorescence. In addition, 2-methylthioadenosine triphosphate (2MeS-ATP, a selective endothelial P2 receptor agonist) was used to infer the selectivity of fura-2 loading. It was reasoned that 2MeS-ATP should produce a decrease in smooth muscle [Ca 2+] i and an increase in endothelial [Ca 2+] i in selectively loaded vessels, consistent with its role as an NO-dependent dilator. In smooth muscle loaded vessels, [Ca 2+] i went from 252±8 to 82±9 nM following luminal administration of 2MeS-ATP, whereas in endothelial loaded vessels, [Ca 2+] i went from 137±11 to 271±20 nM. Thus, a method is provided which allows for selective measurement of endothelial or smooth muscle [Ca 2+] i with simultaneous measurement of diameter in a pressurized cerebral vessel.