Selective measurement of endothelial or smooth muscle [Ca2+](i) in pressurized/perfused cerebral arteries with fura-2

Despite a critical role for calcium in endothelial regulation of cerebrovas cular tone, endothelial intracellular calcium ([Ca2+](i)) has never been me asured in the context of an intact pressurized cerebral vessel. The purpose of the present study was to selectively measure endothelial or smooth musc le [Ca2+](i) and diameter in a pressurized/perfused cerebral vessel. In a p ressurized rat middle cerebral artery, fura-2 AM was administered selective ly to either the luminal (endothelium) or abluminal (smooth muscle) side of the vessel. Selectivity of loading was determined by measuring fura-2 fluo rescence before and after removal of the endothelium. Removal of the endoth elium virtually eliminated fura-2 fluorescence. In addition, 2-methylthioad enosine 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 [Ca2+](i) and an incre ase in endothelial [Ca2+`](i) in selectively loaded vessels, consistent wit h its role as an NO-dependent dilator. In smooth muscle loaded vessels, [Ca 2+](i) event from 252 +/- 8 to 82 +/- 9 nM following luminal administration of 2MeS-ATP, whereas in endothelial loaded vessels, [Ca2+](i) went from 13 7 +/- 11 to 271 +/- 20 nM. Thus, a method is provided which allows for sele ctive measurement of endothelial or smooth muscle [Ca2+](i) with simultaneo us measurement of diameter in a pressurized cerebral vessel. (C) 2000 Elsev ier Science B.V. All rights reserved.