Imaging the nanomolar range of nitric oxide with an amplifier-coupled fluorescent indicator in living cells

Abstract

Nitric oxide (NO) is a small uncharged free radical that is involved in diverse physiological and pathophysiological mechanisms. NO is generated by three isoforms of NO synthase, endothelial, neuronal, and inducible ones. When generated in vascular endothelial cells, NO plays a key role in vascular tone regulation, in particular. Here, we describe an amplifier-coupled fluorescent indicator for NO to visualize physiological nanomolar dynamics of NO in living cells (detection limit of 0.1 nM). This genetically encoded high-sensitive indicator revealed that approximately 1 nM of NO, which is enough to relax blood vessels, is generated in vascular endothelial cells even in the absence of shear stress. The nanomolar range of basal endothelial NO thus revealed appears to be fundamental to vascular homeostasis.

Fig. 1.

An amplifier-coupled fluorescent indicator for visualizing NO in single living cells. (a) Schematic representations of domain structures of sGCα, sGCβ, CGY, sGCα-CGY, and sGCβ-CGY. The amino acid sequence of FLAG tag and linker (Ln) is shown at the bottom. The heterodimer of sGCα-CGY and sGCβ-CGY has been named NOA-1. (b) Principle of the NO indicator NOA-1. sGCα-CGY and sGCβ-CGY are spontaneously associated to form a matured heterodimer, that is, NOA-1. NOA-1 binds with NO and generates cGMP at the rate of 3,000–6,000 molecules per min. Thus generated cGMP binds to the CGY domain in NOA-1 and makes NOA-1 emit a FRET signal. About 99.9% of cGMP molecules thus generated diffusely and bound to NO-free NOA-1. As a result, even a single NO molecule can trigger a large amount of NOA-1 to emit FRET signals. Even if sGCα-CGY and sGCβ-CGY exist as monomers, the monomers also emit FRET signals upon binding with generated cGMP.

Fig. 2.

NOA-1 reversibly sensitizes the nanomolar range of NO in single living cells. (a) Responses of sGCα-CGY (•) and sGCβ-CGY (•) for 50 nM NOC-7 and subsequent 2 mM 8-Br-cGMP stimulation in CHO-K1 cells. (b) Response of the heterodimer of sGCα-CGY and sGCβ-CGY to 5 nM NOC-7 in the absence (black circles) or the presence of 100 μM NS 2028 (blue circles) or 200 μM zaprinast (red circles). (c) Reversible response of NOA-1 for various concentrations of NOC-7. (d) Pseudocolor images of the CFP/YFP emission ratio of NOA-1 before (1,570 s) and after addition of 10 nM NOC-7. (e) Dose–response of NOA-1 for the nanomolar range of NO.

Fig. 3.

Selectivity of NOA-1. (a) Time courses of NOA-1 response for 10 μM CO (red open circle), 100 μM CO (red closed circle), and 5 nM NO (black closed circle) in CHO-K1 cells. (b) Changes in the CFP/YFP emission ratio of NOA-1 upon stimulation with 10 μM CO, 100 μM CO, and 5 nM NO in CHO-K1 cells. (c) Time course of NOA-1 response for 100 nM atrial natriuretic peptide (ANP) or 100 μM isoproterenol in a CHO-K1 cell.

Fig. 4.

Vascular endothelial cells stably generate the nanomolar range of NO. (a) Response of NOA-1 for NO transiently generated upon 1 nM bradykinin and that upon shear stress in vascular endothelial cells. When the cells were pretreated with 1 mM l-NAME, both the response of NOA-1 for bradykinin and that for shear stress disappeared (Insets). (b) Pseudocolor images of the CFP/YFP emission ratio in CHO-K1 and endothelial cells that are expressed with NOA-1. (c) Time course of NOA-1 response in a CHO-K1 cell and an endothelial cell upon 1 mM l-NAME stimulation. (d) Changes in the CFP/YFP emission ratio of NOA-1 upon 1 mM l-NAME stimulation in CHO-K1 and endothelial cells. (e) Time courses of NOA-1 response in CHO-K1 cell (•) and endothelial cell (•) upon 10 μM NOC-7 and subsequent 2 mM 8-Br-cGMP stimulation. (f) Changes in the CFP/YFP emission ratio of NOA-1 in CHO-K1 (•) and endothelial cells (•) upon 10 μM NOC-7 stimulation. (g) Time course of NOA-1 response upon 200 μM zaprinast and subsequent 2 mM 8-Br-cGMP stimulation in an endothelial cell. (h) Changes in the CFP/YFP emission ratio of NOA-1 upon 200 μM zaprinast stimulation in CHO-K1 and endothelial cells. (i) Basal concentration of NO stably generated in a vascular endothelial cell was measured by in situ calibration of NOA-1 for NO. The endothelial cell was treated in advance with 1 mM l-NAME to remove the basal NO, and various concentrations of NO were subsequently added to the endothelial cell. Solutions containing various concentrations of NO were prepared by diluting the saturated NO solution prepared by bubbling NO gas.