Introduction
NO is an important small molecule in the cell, serving as both a direct-response element and an initiator of secondary activities. Numerous proteins that produce or bind NO have been identified, as have proteins and small molecules (cyclic guanosinemonophosphate, or cGMP, in particular) whose production is regulated by NO. The impact of these discoveries has been tremendous, and the 1999 Nobel Prize in Physiology or Medicine recognized pioneering work in the field1, 2, 3. Despite the significance of NO as a cellular player, our knowledge of its behavior is limited. Of particular importance are the following questions: where and when is NO produced? Where is it localized? How long does it persist? The difficulty in answering these questions arises from the limited tools available for determining exactly where and when NO is present. Here, Lippard and co-workers demonstrate the utility of a new turn-on fluorophore for immediate and selective NO detection4.
As NO is a colorless diatomic species, there have been no viable methods for its direct intracellular detection and thus all work on this molecule so far has necessarily relied on indirect detection or inference. In the early stages of the field, researchers made observations based on indirect responses by, for example, controlling expression of NO synthases (NOS), following the NO-dependent formation of cGMP and observing cellular function in the presence of NO gas1, 2, 3, 5. Electron paramagnetic resonance (EPR) has been successfully applied to image NO in organs and even whole organisms. However, spatial resolution of EPR is on the order of 1 cm, precluding its use for imaging at the cellular level.
Fluorescence microscopy techniques have the appropriate spatial resolution (micron) and timescale (millisecond) for imaging cellular processes in real time. As a result, attention has turned to the development and application of NO-responsive fluorescent probes: small molecules or protein constructs that translate the presence of NO to a visible fluorescence signal5. The earliest fluorescent probes for NO were based on the reaction of o-diamine derivatives (Fig. 1a)6. Though effective, such probes do not react with NO directly but rather with the nitrite anion (NO2–) produced by the reaction of NO and O2. This is problematic in that the production of nitrite can be slow at low O2 concentrations, limiting both the spatial and temporal resolution of such probes. More recently, a fluorescent fusion protein for NO imaging has been reported in which NO induces cGMP formation and the cGMP turns on a fluorescence response7. Although this probe has the advantage of being genetically encodable, it too suffers from the limitation of imaging a diffusible non-NO species rather than NO itself.
Figure 1: Small-molecule probes for fluorescence imaging of intracellular NO.
(a) A near-infrared emissive NO-imaging agent. (b) The fluorescein conjugate described by Lim et al. turns on in response to NO-mediated reduction of Cu(II).
Full size image (47 KB)There have been several recent reports of NO-responsive fluorophores in which the fluorescence emission is regulated by the reaction of NO with Cu(II)-quenched fluorophore complexes in which NO reduces Cu(II) to Cu(I)7, 8, 9, 10. The two oxidation states of copper have very different preferred coordination geometries; thus a change in oxidation state leads to release of Cu(I) from the complex and 'unquenching' of the emission. The current study by Lim et al. reports the first practical application of this approach for the cellular imaging of NO concentrations (Fig. 1b). The probe molecule is a derivative of fluorescein with a Cu(II)-chelating group attached. On reaction with NO, Cu(II) is reduced to Cu(I) and released, with concomitant nitrosation of the chelating group. The initial Cu(II) complex is essentially nonfluorescent, whereas the nitrosated copper-free species is brightly fluorescent. The reaction with NO is rapid and occurs only with NO, not with by-products of other NO reactions. The excitation and emission wavelengths are compatible with standard fluorescein optical filters, and the limit of detection is on the order of 1 nM NO. The authors provide a detailed mechanistic analysis of the NO-induced transformation in vitro, and they show in vivo that it can indeed be used to image cellular NO and (just as important) that neither the nitrosated fluorophore nor the small amount of Cu(I) released is significantly toxic.
This new compound represents an alternative to protein constructs and reactive organic agents, which have inherent disadvantages. Though some important challenges (such as exact response time and quantification of NO concentration in absolute terms) still need to be addressed, this small molecule is a very promising tool for NO imaging and is likely to have a substantial impact on the study of intracellular NO synthesis, localization and persistence. Perhaps equally important, it also sets the stage for the next essential advance in small-molecule NO imaging of reversible binding and signaling, a process that would enable detection of both increases and decreases in NO concentration in real time.
