Effect of nitric oxide on naphthoquinone toxicity in endothelial cells: role of bioenergetic dysfunction and poly (ADP-ribose) polymerase activation

Abstract

When produced at physiological levels, reactive oxygen species (ROS) can act as signaling molecules to regulate normal vascular function. Produced under pathological conditions, ROS can contribute to the oxidative damage of cellular components (e.g., DNA and proteins) and trigger cell death. Moreover, the reaction of superoxide with nitric oxide (NO) produces the strong oxidant peroxynitrite and decreases NO bioavailability, both of which may contribute to activation of cell death pathways. The effects of ROS generated from the 1,4-naphthoquinones alone and in combination with NO on the activation status of poly(ADP-ribose) polymerase (PARP) and cell viability were examined. Treatment with redox cycling quinones activates PARP, and this stimulatory effect is attenuated in the presence of NO. Mitochondria play a central role in cell death signaling pathways and are a target of oxidants. We show that simultaneous exposure of endothelial cells to NO and ROS results in mitochondrial dysfunction, ATP and NAD(+) depletion, and cell death. Alone, NO and ROS have only minor effects on cellular bioenergetics. Further, PARP inhibition does not attenuate reduced cell viability or mitochondrial dysfunction. These results show that concomitant exposure to NO and ROS impairs energy metabolism and triggers PARP-independent cell death. While superoxide-mediated PARP activation is attenuated in the presence of NO, PARP inhibition does not modify the loss of mitochondrial function or adenine and pyridine nucleotide pools and subsequent bioenergetic dysfunction. These findings suggest that the mechanisms by which ROS and NO induce endothelial cell death are closely linked to the maintenance of mitochondrial function and not overactivation of PARP.

Figure 1. NO abrogates quinone-dependent PARP activation

BAEC were exposed to Deta/NO (500 µM) for 1 h prior to treatment with menadione (20 µM) or DMNQ (20 µM) for an additional 4 h. (A) Protein poly(ADP-ribosyl)ation was measured by Western blotting using antibody against poly(ADP-ribose). (B) Densitometric analysis of protein PAR normalized to β-actin levels. NAD+ (C) and NADH (D) levels were measured by HPLC and normalized to total protein. Control (black bars), menadione-treated (white bars), and DMNQ-treated (grey bars) groups are shown. Values represent means ± SE; n = 3. * p < 0.05 compared to untreated control, ** p < 0.05 compared to samples without Deta/NO.

Figure 2. NO and quinone synergistically contribute to cell death

BAEC were exposed to Deta/NO (500 µM) for 1 h prior to treatment with menadione (20 µM) or DMNQ (20 µM) for an additional 4h. (A) Light micrographs were taken at 5 h and show morphological changes in cells exposed to both quinone and NO donor. (B) Viability was assessed by MTT in cells incubated in the absence or presence of PJ-34 (10 µM). (C) Cells were washed and incubated in full medium for an additional 12 h. LDH release was measured to assess the cell viability. Control (black bars), menadione-treated (white bars), and DMNQ-treated (grey bars) groups are shown. Values represent means ± SE; n = 3. * p < 0.05 compared to samples without Deta/NO, **p < 0.05 compared to samples without PJ-34.

Figure 3. The effect of PARP inhibitor on NAD⁺ depletion

BAEC were exposed to Deta/NO (500 µM) for 1 h prior to incubation without (black bars) and with menadione (20 µM, white bars) for an additional 4 h in the presence and absence of PARP-1 inhibitor, PJ-34 (10 µM). NAD+ levels were measured by HPLC and normalized to total protein. Values represent means ± SE; n = 3. * p < 0.05 compared to samples without Deta/NO, **p < 0.05 compared to samples without PJ-34.

Figure 4. Changes in adenine nucleotides in response to combination of NO and quinone

BAEC were exposed to Deta/NO (500 µM) for 1 h prior to treatment with menadione (20 µM, white bars) or DMNQ (20 µM, grey bars) for an additional 4 h. Black bars represent control treatment. ATP (A), ADP (B) and AMP (C) levels were measured by HPLC and normalized to total protein. Values represent means ± SE; n = 3. * p < 0.05 compared to samples without Deta/NO.

Figure 5. Mitochondrial function is decreased in response to combination of NO and quinone

BAEC were exposed to Deta/NO (500 µM) for 1 h prior to treatment with menadione (20 µM, white bars) or DMNQ (20 µM, grey bars) for an additional 4 h. Black bars represent control treatment. (A) Basal oxygen consumption rate (OCR), (B) ATP-linked OCR and (C) reserve capacity OCR were measured using extracellular flux technology. OCRs were normalized to total protein per well after completion of assay. Values represent means ± SE; n = 3 to 4. * p < 0.05 compared to control, ** p < 0.05 compared to samples without Deta/NO.