Real-time visualization of distinct nitric oxide generation of nitric oxide synthase isoforms in single cells

Figures

• 

  Fig. 1. Simultaneous imaging of Ca2+ and NO in single endothelial cells. (A) The scheme represents the functional principle of G-geNOp that FP fluorescence is quenched upon NO binding to the probe. (B) Representative wide field images of EA.hy926 cells expressing G-geNOp prior to exposure (left panel) and in the presence of the NO-donor NOC-7 (10 μM, right panel). (C) Representative [Ca2+]cyto (black curve) and [NO]cyto (blue curve) signals shown in percentage of the respective maximal increases in response to 100 μM histamine. Curves represent at least 4 independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

• 

  Fig. 2. Western Blot analysis of nNOS and eNOS expression in HEK-cell clones (A) Bars represent the expression rate of eNOS (blue bars) and nNOS (green bars) in wild-type (left panel, n = 3), eNOS- (middle bars, n = 3) and nNOS-expressing HEK293 cells (right bars, n = 3). (B) Representative Western blot of total homogenates of wild-type, eNOS- or nNOS-expressing HEK293 cells (10 μg of protein) probed for eNOS (140 kDa), nNOS (155 kDa) and β-actin (43 kDa) expression (n = 3). Mean values are shown ±SD, *P < 0.05 versus control using unpaired t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

• 

  Fig. 3. Ca2+-triggered eNOS- and nNOS-specific NO signals. (A) Representative time course of cytosolic Ca2+ (upper panel) and NO signals (lower panel) in response to 100 μM ATP in wild-type HEK293 cells (left panel), stably eNOS-expressing HEK293 cells (middle panel), and stably nNOS-expressing HEK293 cells (right panel). NO recordings were performed using C-geNOp. (B) Bars represent mean values in ±SD of basal fura2 ratios (left panel) and maximum cytosolic Ca2+ elevations in response to 100 μM ATP (right panel) of wild-type HEK293 cells (black bars, n = 52/12), eNOS expressing HEK293 cells (blue bars, n = 53/12) and nNOS expressing HEK293 cells (green bars, n = 65/12). (C) Representative pulsatile NO signals in nNOS-expressing HEK cells (D) Overlay of representative cytosolic Ca2+ (black curve) and NO recordings of stably nNOS- (green curve) or eNOS- (blue curve) expressing HEK293. Experiments were at least performed 4 times. (E) Single cell NO responses (light grey curves) and mean curves of eNOS-expressing HEK cells (blue curve, n = 5/32) and nNOS-expressing HEK cells (green curve, n = 5/37). (F) Bars represent the half time (t1/2) of maximum fura-2 ratio (upper blue bar, n = 54/4…

• 

  Fig. 4. pH sensitivity of geNOps. (A) Representative time course of cytosolic pH changes in response to 100 μM ATP in eNOS-HEK cells (left panel) and nNOS-HEK cells (right panel) expressing the cytosolic SypHer. (B) Bars represent mean values in ±SD of maximal sypHer ratio changes in response to 100 μM ATP of eNOS-HEK cells (blue bars, n = 4/41) and nNOS-HEK cells (green bars, n = 4/46). (C) Representative time course of NO signals of eNOS-HEK cells (left panel) and nNOS-HEK cells (right panel) to 100 μM ATP. Cells were transiently transfected with the NO insensitive geNOpmut. (D) Bars represent mean values in ±SD of maximal geNOpmut responses upon administration of 100 μM ATP of eNOS-HEK cells (blue bars,n = 4/21) and nNOS-HEK cells (green bars, n = 4/21). Mean values are shown ±SD, *P < 0.05 versus control using unpaired t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

• 

  Fig. 5. Ca2+-evoked nNOS- and eNOS-derived NO generation profiles in ionomycin permeabilized cells (A) Mean curves represent NO generation profiles derived from eNOS (blue curve) and nNOS (green curve) in response to Ca2+ addition. Experiments were performed in the presence of 5 μM ionomycin. NO recordings were performed using C-geNOp. (B) Bars represent respective statistics of panel A, maximum initial slope of nNOS- (green bar, n = 34/3) and eNOS-derived (blue bar, n = 36/3) NO signals. Mean values are shown ±SD, *P < 0.05 versus control using unpaired t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

• 

  Fig. 6. CR-geNOp, a ratiometric NO probe based on two FPs. (A) Schematic structures of CR-geNOp consisting of the cyan FP (CFP), a NO-sensitive GAF domain, a rigid linker (EAAAK)n and the red FP (tagRFP) in the presence and absence of NO, which quenches CFP fluorescence only (B) Left panel: fluorescence intensity CFP (cyan curve) and RFP (red curve) over time in cells expressing CR-geNOp. Cells were treated with 10 μM NOC-7 as indicated. Right panel: respective ratio signal (red/cyan; F584/F480) of CR-geNOp (n = 98/5). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

• 

  Fig. 7. Visualizing of NO dynamics in iNOS-expressing cells (A) Representative pseudo-colored ratio images (F584/F480) of CR-geNOp-transfected wild-type (upper images) and iNOS-expressing HEK293 cells (lower images) under initial conditions (left panel) and in the presence of 500 μM L-NNA (middle panel) or 10 μM NOC-7 (right panel). Scale bar represents 10 μm. (B) Mean curves (bold line) and single cell traces (thin lines) showing NO signals of stably iNOS-expressing (red bold curve, n = 23/3) and wild-type HEK293 cells (black bold curve, n = 15/3). Cells were treated with 300 μM L-NNA followed by administration of 10 μM NOC-7. (C) Representative time course showing NO signals in arginine-depleted (red curve) versus non-depleted iNOS-expressing HEK293 cells (black curve). Cells were treated with 50 μM L-arginine and 500 μM L-NNA as indicated. Bars represent respective statistics from panel C. Maximal ratio changes of arginine depleted iNOS-expressing (red bar, n = 21/3) and non-depleted iNOS HEK293 cells (black bar, n = 7/3) upon 50 μM L-arginine supplementation. Mean values are shown in ±SD, *P < 0.05 versus control using unpaired t-test. (D) Correlation of maximal arginine-induced…