In 1998 R.F. Furchgott, L.J. Ignarro, and F. Murad received the Nobel Prize in Physiology and Medicine for their discoveries concerning nitric oxide (NO) as a signaling molecule in the cardiovascular system. This indicates the importance the scientific community has placed on the role of nitric oxide as a signal transporter in neurons, endothelial cells, and in the immune system. NO has been implicated in vasodilation,1 neurotransmission,2 cytotoxicity, immune response,3 and inflammation.4-6
Within cells, nitric oxide synthase (NOS) catalyzes the conversion of arginine to citrulline plus NO in the presence of molecular oxygen, tetrahydrobiopterin, NADPH, and flavin cofactors. Nε-hydroxy-L-arginine is an intermediate in this reaction. NO has an in vivo half-life of only a few seconds;7 however, since it is soluble in both aqueous and lipophilic media, it readily diffuses through the cytoplasm and across cell membranes. NO has effects on neuronal transmission as well as synaptic plasticity in the central nervous system. NO activates guanylate cyclase in the vasculature, thus, increasing the synthesis of cGMP, which in turn induces smooth muscle relaxation and vasodilation. NO toxicity is related to its ability to combine with superoxide anions to form peroxynitrite, an oxidizing free radical that can damage DNA and other cellular constituents.
The NO molecule is unstable and occurs in very low concentrations in biological systems. In order to detect it in real time, methods with high sensitivities and specificities are required. Fluorescence microscopy seems to be the method of choice, provided suitable NO probes are available. This high spatial-temporal resolution technique allows for reliable imaging of in situ NO concentrations.
Research into the design of sensitive NO probes revealed a reaction involving vicinal aromatic diamino compounds8,9 that allow for selective trapping and detection of NO. Figure 1 illustrates this reaction using the novel NO probe 5,6-diaminofluorescein diacetate (DAF-2-DA) as an example.
Figure 1.Principle of intracellular NO measurement, shown with the probe DAF-2 DA. Lipophilic, non-fluorescent DAF-2-DA permeates the cell membrane and is then hydrolyzed by cytosolic esterases to the weakly fluorescent probe DAF-2. The presence of NO radicals transforms it to the strongly fluorescent triazole DAF-2 T.
2,3-Diaminonaphthalene (DAN) was established as a sensitive probe to measure NO-concentration in vitro and under physiological conditions.11 The respective 2,3-naphthotriazole (NAT) was detected in the low nM concentration range. DAN, however, proved to be ill-suited to NO detection in cells due to rapid leakage through cell membranes after loading. High background fluorescence in biological matrices was also experienced due to its rather short excitation and emission wavelengths.
Therefore, DAN-1 and its cell membrane permeable precursor DAN-1-EE were developed.12DAN-1 is formed by the hydrolysis of DAN-1-EE by cytosolic esterases. DAN-1-T (Figure 2), the fluorescent reaction product of DAN-1 with NO, showed considerably improved leakage behavior.
Figure 2.Time course of formation of fluorescent DAN-1-T from DAN-1 upon addition of final concentration of 5 mM (green line) or 50 mM (red line) respectively of a NO forming agent (NONOate).
Fluorescein and rhodamine-based probes have been developed in order to eliminate some drawbacks of naphthalene-based NO probes, such as cytotoxicity, strong autofluorescence, a small extinction coefficient, poor solubility in neutral buffer, and short excitation/emisson wavelengths.10,13-16
Limit of detection (LOD) of NO by DAF-2 is 5 nM in vitro, in the absence of absorbing side products. Oxidized forms of NO such as NO2- and NO3-, as well as reactive oxygen species such as O2 ·-,H2O2, and ONOO- do not react with DAF-2 to give a fluorescent product. Therefore, under physiological conditions fluorescent DAF-2 T is only formed in the presence of NO.
A fluorescent response was detected when cultured, smooth aortic rat muscle cells loaded with DAF-2-DA were viewed using a confocal laser scanning microscope in the presence of endotoxins and cytokines. The addition of L-arginine resulted in a sharp increase of the fluorescence signal, whereas supplementary addition of N-monomethyl-L-arginine (L-NMMA) resulted in no signal increase, clearly demonstating the inhibition of NO-synthase.
The fluorescein type probe (DAF-2) shows a bright fluorescence signal, while the rhodamine type probes (DAR-1 and DAR-2) excel with high photostability and their applicability over a broad pH range (Table 1, Table 2 and Figure 3).
Figure 3.pH dependence of fluorescence intensity (a.u.) of DAF-2 (green), DAR-1 (orange) and DAR-2 (pink).
The detection of molecular oxygen plays an important role in many applications and biological processes. The probes are oxygen-sensitive indicators that change their fluorescense intensity or emission maximum after binding to molecular oxygen. The sensors are reversible and are stable against photobleaching.
Reactive oxygen probes (ROS) are associated with research in many diseases like inflammatory and cancer, where increased levels of ROS are produced in cells. The study of ROS requires photostable sensors with minimal autooxidation and low production rates of by-products.17