Parsing the Effects of Nitric Oxide, S-Nitrosothiols, and Peroxynitrite on Inducible Nitric Oxide Synthase–Dependent Cardiac Myocyte Apoptosis
The actions of nitric oxide (NO) mediating vasorelaxation through activating guanylate cyclase to produce cGMP are now well-established, as recognized by the award of the Nobel prize in 1998. However, the production of NO is also implicated in a much broader range of physiological and pathological actions that are independent of cGMP. Cytokines in particular are known to greatly stimulate NO production as part of the nonspecific immune system and may play a significant role in heart disease. Cytokines are known to induce expression of the inducible nitric oxide synthase (iNOS) in cardiac myocytes, which contributes in part to myocyte contractile dysfunction.
In this issue of Circulation Research, Arstall et al1 demonstrate that iNOS upregulation by interleukin-1β and interferon-γ increases apoptosis in cultured myocytes by a process that was independent of guanylate cyclase activation and cGMP. Cell death was blocked by both selective and general iNOS inhibitors, clearly implicating the production of NO in cell death. However, concentrations of an NO donor added in similar concentrations to the amount of endogenous nitrite produced by myocytes after cytokine treatment did not stimulate cell death, suggesting that NO itself was not cytotoxic.
The chemical reactivity and toxicity of NO can be greatly increased by its diffusion-limited reaction with superoxide (O2.−) to form peroxynitrite (ONOO−). A role for ONOO− was implicated in myocyte apoptosis by Arstall et al1 by showing that the O2.−and ONOO− scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) protected myocytes from cytokine-induced apoptosis. Myocytes were also shown to be very sensitive to ONOO−, with increased cell death becoming apparent with as little as 10 μmol/L bolus additions. ONOO− has been shown previously to decrease myocardial contractility in cultured myocytes.2
An intriguing aspect of the present study was the treatment of rat cardiac microvascular endothelial cells with cytokines, which did not induce endothelial cell death in spite of upregulated iNOS expression. However, NO produced by the endothelium was able to diffuse to ventricular myocytes in mixed cultures and induce apoptosis. This suggests that myocytes under these conditions were producing O2.−, which made endothelial production of NO toxic in a paracrine fashion.
The comparison of toxicity with different NO donors as well as NO-derived oxidants requires careful consideration. Arstall et al1 found that myocytes survived treatment with the NO donor molecule, 100 μmol/L S-nitrosocysteine, but 10-fold higher concentrations did increase apoptosis to ≈33% after 48 hours of treatment. In contrast, 100 μmol/L ONOO− caused 49% apoptosis. The difference in toxicity becomes far more dramatic when the short half-life of ONOO− is taken into account. At neutral pH, the half-life of ONOO− is under 1 second, depending on the temperature and components in the buffer. Consequently, most of the ONOO− decomposed before reaching the myocytes and then was gone after a few seconds. The rate of S-nitrosocysteine decomposition depends strongly on the medium and incubation conditions, but can persist for hours. Consequently, the net exposure of myocytes to S-nitrosothiols as measured by the area under the curve of time versus concentration was vastly greater than the net exposure to ONOO−.
There is substantial controversy concerning the proapoptotic versus antiapoptotic effects of NO. A significant source of the controversy results from the type of NO donor used in vitro to produce NO. Many studies use only nitrosothiols as a source of NO. However, the release of NO requires a chemical reduction by one electron.3 NO itself does not react spontaneously with thiols to produce nitrosothiols, because there is an oxidation by one electron required. Although nitrosothiols are widely used as NO donors, it is critical to recognize that nitrosothiols have their own chemical reactivities distinct from NO, and their actions cannot be simply equated with NO. Nitrosothiols are themselves far more reactive with biological thiols than NO, producing multiple products including nitrosation, thiolation, and can leave sulfur in higher oxidation states. Consequently, nitrosothiols cannot be equated with NO.
For example, treatment of cells with nitrosothiol donors can inhibit apoptosis at least in part by S-nitrosation of the active site caspases.4 5 6 In the present study, the apparent lack of toxicity of 100 μmol/L S-nitrosocysteine may be in part due to inhibiting caspases whereas other actions may be stimulating proapoptotic cascades.
Other NO donors can directly release NO. Consequently, the toxicity of NO itself can be tested experimentally by using low concentrations of the long-lived NO donor DETA-NONOate. This compound spontaneously decomposes to release two NO molecules with a half-life of ≈50 hours in cell culture medium. Adding 20 μmol/L DETA-NONOate will maintain an 80 to 100 nmol/L steady-state concentration of NO in cell culture medium for at least 48 hours.7 Depending on the cell culture medium, it may be useful to add 10 U of superoxide dismutase per milliliter to scavenge the slow flux of O2.− produced by cell culture medium, which will increase the steady-state concentration of NO by ≈30%. NO at low concentrations in the absence of O2.− is very stable, because its reaction with oxygen is extremely slow. The decomposition of NO by oxygen increases with the square of NO concentration, so maintaining a steady-state concentration of 1 μmol/L NO would require adding 400 μmol/L DETA-NONOate. However, exposure to 100 nmol/L NO for 48 hours gives an immense exposure to NO, as measured by the area under the curve of concentration versus time. A simple comparison of the different types of NO donors with consideration of the time of exposure could help unravel the vast conflicting literature on the actions of NO donors.
In the present study, inhibition of NO production strongly protected myocytes from apoptosis. One can get a sense of how little NO might be required to be toxic to cells by assessing what steady-state concentration of NO generated from DETA-NONOate is necessary to restore toxicity. In cultured motor neurons subjected to trophic factor deprivation, 100 nmol/L NO overcame the protection provided by NOS inhibitors and increased tyrosine nitration.7 However, the same concentration of NO was not toxic and even promoted survival of motor neurons treated with trophic factors and did not cause tyrosine nitration.8 These considerations illustrate how the actions of NO may begin to be separated from those of ONOO− and other oxidative products derived from NO.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- © 1999 American Heart Association, Inc.
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