Selective measurement of endothelial or smooth muscle [Ca(2+)](i) in pressurized/perfused cerebral arteries with fura-2.
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.
Marrelli
S P
SP
Department of Anesthesiology, Baylor College of Medicine, Houston, TX 77030, USA. sm691025@bcm.tmc.edu
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D016477:Artifacts; D002118:Calcium / Q000378:metabolism*; D002560:Cerebrovascular Circulation / Q000502:physiology; D004730:Endothelium, Vascular / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2; D008297:Male; D020768:Middle Cerebral Artery / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000378:metabolism*; D009482:Neurophysiology / Q000295:instrumentation / Q000379:methods*; D009569:Nitric Oxide / Q000378:metabolism; D010477:Perfusion; D011312:Pressure; D051381:Rats; D020318:Rats, Long-Evans; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology; D014466:Ultraviolet Rays; D014661:Vasoconstriction / Q000502:physiology / Q000528:radiation effects
D005456:Fluorescent Dyes; D018048:Receptors, Purinergic P2; D013873:Thionucleotides; D009569:Nitric Oxide; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2; C008501:2-methylthio-ATP