Simultaneous measurement of intracellular Ca2+ and nitric oxide: a new method

Different methods to measure the unstable radical nitric oxide (NO) have be en established. We are going to present a new method to measure intracellul ar calcium and NO simultaneously in endothelial cells. A new fluorescent dy e (DAF-2) has been developed recently which binds NO resulting in an enhanc ed fluorescence. We loaded porcine aortic endothelial cells with Fura-2, a fluorescent dye commonly used to measure intracellular calcium, and DAF-2 s imultaneously (cell permeable dyes). Using excitation wavelengths of lambda 340 nm (Fura-2) and lambda 485 nm (DAF-2) we could show that thrombin indu ces an intracellular calcium increase and simultaneously a NO formation in endothelial cells which could be blocked by a NO synthase inhibitor. This n ew method of a simultaneous measurement of intracellular calcium and NO pro vides the possibility to follow intracellular calcium and NO distributions online, and is sensitive enough to monitor changes of NO formed by the cons titutive endothelial NO-synthase. (C) Harcourt Publishers Ltd 2000.