Nitric oxide (NO) is a small, biologically active messenger molecule that has many important roles in human physiology ranging from homeostasis to pathology. NO acts as a potent vasodilator, inhibits platelet aggregation, reduces platelet adherence to the endothelium and suppresses vascular smooth muscle proliferation1. NO derived from a constitutive NO synthase in the brain is an important neurotransmitter that participates in the genesis of memory and serves as a mediator of the metabolism/blood flow coupling in the brain. It also plays an important role in neuromorphogenesis and synaptic plasticity. A reduction in vascular NO plays a significant role in the development of atherosclerosis, the pathophysiology of diabetes, subarachnoid hemorrhage, ischemia-reperfusion injury and shock2,3. On the other hand, enhanced formation of NO in the brain plays a role in neuronal injury during stroke, acute and chronic neurodegeneration, and epileptic seizures4,5.
Studies on the physiological roles of NO have been hampered by the difficulty in direct in vivo measurements of this compound due to its extremely short half-life and low concentrations. A new dye, 4,5-Diaminofluorescein Diacetate (DAF-2 DA), can be used for real-time bioimaging of NO with fine temporal and spatial resolution6. Cells are loaded with the membrane-permeable DAF-2 DA which is subsequently hydrolyzed by cytosolic esterases releasing free DAF-2 that does not leak into the medium. At physiological pH, the DAF-2 is relatively non-fluorescent, however, in the presence of NO and oxygen, a DAF-2 triazole (DAF-2T), a fluorescent product, is formed.
This conversion of DAF-2 to DAF-2T by reaction with NO causes little change in the absorbance maximum, but increases the quantum efficiency by over 180-fold. In several recent studies, this dye has been used to detect NO production in LPS stimulated macrophages7 and cytokine activated rat aortic smooth muscle cells6,8. The increase in fluorescence intensity was positively correlated to the concentrations of LPS and cytokines used. The fluorescence intensity was significantly reduced by the NO synthase inhibitor L-NMMA. The sensitivity of DAF-2 is such that an increase in fluorescence intensity was observed even in resting endothelial cells, reflecting the basal NO production7. In vitro studies have shown that DAF-2 does not react with stable oxidized forms of NO such as NO2-, NO3-, nor with other reactive oxygen species such as O2.-, H2O2, and ONOO-8. Other advantages of using DAF-2 for cellular imaging are its visible excitation wavelength that is less damaging to cells, and the fact that it is not subject to interference from the autofluorescence of biological samples.
For more information: Margaret Dentliner, sr. product manager and marketing director, CalBiochem Novabiochem, telephone: 619-450-5569.