A Mitochondria-Specific Fluorescent Probe for Visualizing Endogenous Hydrogen Cyanide Fluctuations in Neurons

Abstract

An ability to visualize HCN in mitochondria in real time may permit additional insights into the critical toxicological and physiological roles this classic toxin plays in living organisms. Herein, we report a mitochondria-specific coumarin pyrrolidinium-derived fluorescence probe (MRP1) that permits the real-time ratiometric imaging of HCN in living cells. The response is specific, sensitive (detection limit is ca. 65.6 nM), rapid (within 1 s), and reversible. Probe MRP1 contains a benzyl chloride subunit designed to enhance retention within the mitochondria under conditions where the mitochondria membrane potential is eliminated. It has proved effective in visualizing different concentrations of exogenous HCN in the mitochondria of HepG2 cells, as well as the imaging of endogenous HCN in the mitochondria of PC12 cells and within neurons. Fluctuations in HCN levels arising from the intracellular generation of HCN could be readily detected.

Figure 1.

(a) Changes in the fluorescence emission spectra of MRP1 (1 μM) seen upon the addition of increasing quantities of cyanide (0− 60 μM as its KCN salt) at room temperature (λ_ex = 467 nm). (b) Time-dependent changes in the fluorescence emission intensities recorded at 490 and 599 nm observed upon treatment of MRP1 (1 μM) with cyanide (60 μM). (c) Changes in the fluorescence intensity of MRP1 (1 μM) seen upon the sequential addition of first cyanide (60 μM) and then AgNO₃ (60 μM) (λ_ex = 467 nm, λ_em = 599 nm); (■) MRP1, (blue ▲) MRP1 + CN⁻, and (red ●) MRP1 + CN⁻ + AgNO₃. (d) Ratiometric fluorescence response of MRP1 (1 μM) seen upon exposure to 60 μM of various putative analytes. Inset: Photos of MRP1 (1 μM) taken in the presence of various species (60 μM) under the illumination of a hand-held UV lamp. All studies were carried out in 20 mM potassium phosphate buffer/acetonitrile, pH 7.4, 1:3 v/v.

Figure 2.

Confocal image of HepG2 cells costained with either MRP1 and MTDR or 1b and MTDR. (a−c) Bright field, red (λ_em = 575−650 nm), and NIR (λ_em = 660−740 nm) images of HepG2 cells costained with MRP1 and MTDR. (d) Intensity profile of regions of interest (ROIs) in the costained HepG2 cells as indicated by the white arrows in (b) and (c). (e−g) Bright field, red, and NIR images of HepG2 cells costained with 1b and MTDR. (h) Intensity profile of the ROIs indicated in (f) and (g).

Figure 3.

Confocal images of HepG2 cells stained with MRP1 (0.5 μM) or 1b (0.5 μM) in the presence of CCCP (10 μM) and recorded at different time intervals. The images were produced by monitoring the red channel (λ_em = 575−650 nm).

Figure 4.

Confocal images of live HepG2 cells stained with MRP1 (0.5 μM) in the presence of different concentrations of exogenous cyanide. (a−c) Images recorded over the green (λ_em = 460−530 nm) and red (λ_em = 575−650 nm) channels, and the corresponding (F_green/F_red) ratio for HepG2 cells stained with MRP1 cells and incubated with 0 μM cyanide. (d−f) Green and red channel and ratiometric imaging of HepG2 cells stained with MRP1 and incubated with 15 μM cyanide. (g−i) Green and red channels and ratiometric imaging of MRP1 stained HepG2 cells incubated with 40 μM cyanide. (j−l) Green and red channels and ratiometric imaging of MRP1 stained HepG2 cells pretreated with 40 μM MHb, and then incubated with 40 μM cyanide. (m) Average intensity ratios from images (c), (f), (i), and (l). (n) Linear relationship between the average intensity ratio and cyanide concentration. Error bars represent the standard deviation.

Figure 5.

Confocal image of endogenous mitochondrial HCN in PC12 cells. (a−c) Green (λ_em = 460−530 nm), red (λ_em = 575−650 nm), and ratiometric (F_green/F_red) imaging of PC12 cells incubated with MRP1 (0.5 μM). (d−f) Green, red, and ratiometric imaging of PC12 cells incubated with MRP1 (0.5 μM), and then incubated with 10 μM HYDROM. (g−i) Green, red, and ratiometric imaging of PC12 cells pretreated with 40 μM MHb and then incubated with MRP1 (0.5 μM). (j) Average intensity ratios from images (c), (f), and (i). Error bars represent the standard deviations.

Figure 6.

Confocal image of endogenous mitochondrial HCN in neurons. (a) Bright field image. (b) Ratiometric (F_green/F_red) imaging of the neuron cells after incubation with MRP1 (0.5 μM) for 10 min. (c) Ratiometric (F_green/F_red) imaging of the cells in panel (b) and then further stimulated with HYDROM (10 μM) for 10 min. (d) Ratiometric imaging of the cells in panel (c) that were then treated with MHb (40 μM).

Scheme 1.

(a) Components Making Up the Present Series of HCN Probes;^a and (b) Proposed Basis for the Sensing Response of MRP1 and Related HCN Probes ^aNote that different substituents are present at the 4-position of the phenyl group.