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COMMENTARY
EB2003 SYMPOSIUM
Mitochondrial Nitric Oxide
1 Renal Electrolyte Division University of Pittsburgh School of Medicine Pittsburgh, PA 15261 (E-mail: ajk5{at}pitt.edu)
2 Department of Chemistry Carnegie Mellon University Pittsburgh, PA 15261
THIS SERIES OF SIX PAPERS covers the presentations from the Experimental Biology 2003 Symposium entitled "The Function and Regulation of Mitochondrially Produced Nitric Oxide in Cardiomyocytes," sponsored by the American Physiological Society. Broadly, the topics include views on nitric oxide (NO) biochemistry in the mitochondrion and mitochondria-rich tissues, whether this NO has been endogenously produced by mitochondrial NO synthase (NOS) (mtNOS), or other sources external to the mitochondrion. This collection of papers begins with evidence for the existence of mtNOS (6), moves through considerations of protein nitration (1, 5), explores possible NO signaling pathways (4), and examines the bioavailability of NO (8). The series then concludes with some final comments on NO catabolism (11).
The existence of mtNOS has proven challenge to demonstrate. The possible contamination of mitochondrial preparations with adventitious NOS from other sources is a serious difficulty. Most laboratories have employed an immunostaining approach. However, of the half a dozen or so "specific" antibodies that we have tried, all exhibited cross-reactivity with multiple NOS isoforms, leading to false positive results being the rule rather than the exception in our own laboratories. The problem is exacerbated in cardiomyocytes by the presence of an endothelial NOS (eNOS) isoform located in the caveolae on the inner face of the sarcoplasmic membrane (cardiac eNOS). Consequently, the findings that mtNOS activity is observed only in tightly coupled heart mitochondria and also in mutant cardiomyocytes in which cardiac eNOS is inactive (7) are important results in relation to the contamination issue.
Interestingly, the same neuronal NOS isoform appears to be present in both the mitochondria (7) and the sarcoplasmic reticulum (14). Given the close association of these two organelles, it is not entirely clear whether they both contain the same NOS or if preparations of one can be contaminated by NOS from the other. At present, we have neither a vested interest nor a particular opinion regarding the presence of a NOS in the sarcoplasmic reticulum (srNOS), but we can state with some certainty that mtNOS is not an srNOS-derived contaminant. Structures clearly identified microscopically as mitochondria are associated with the measured mtNOS activity, which ceases when they are uncoupled. Preparations of sarcoplasmic reticulum, on the other hand, tend to be vesicles derived from fragmented membranes.
The existence of mtNOS has started to become more widely accepted and consequently, some colleagues have begun to turn their attention to its possible function(s). Indeed, a number of the papers within this symposium address this intriguing issue. However, current experience suggests that the function(s) of mtNOS may well prove to be subtle. We note with regard to this point that the heart-to-body mass ratios of our mtNOS-deficient mice are indistinguishable from those of wild-type animals.
The first paper by Kanai et al. (6) demonstrates the presence of mtNOS in two diverse cell types, cardiomyocytes and urothelial cells isolated from the urinary bladder epithelium. This finding raises the possibility that mitochondrial NO production may be important in many different cell types. Data are presented suggesting that mtNOS may be protective in one case (cardiomyocytes from a hypertrophied heart) but detrimental in another (urothelial cells exposed to ionizing radiation). The identification of cardiac mtNOS was demonstrated directly by microsensor measurements of NO production from isolated wild-type mitochondria and the absence of this production in mitochondria from neuronal NOS
knockout mice. On the other hand, the isolation of mitochondria from urothelial cells is impractical due to the limited tissue, and so urothelial mtNOS was demonstrated indirectly by a combination of biochemical and electrophysiological approaches, including the first practical application of a peroxynitrite microsensor.
The laboratories of Guilivi (5) and and Stuehr (1) indicate that NO is a precursor of reactive nitrating species such as peroxynitrite (ONOO–) that can modify protein tyrosines to generate 3-nitrotyrosine. Many diseases are associated with increased levels of protein-bound nitrotyrosine, and thus this is used as a marker for oxidative damage (12). Alternative findings are presented here from two laboratories indicating that the nitration of proteins may constitute a posttranslational modification by which NO exhibits long-term bioregulatory effects mediated through interactions with the mitochondrial respiratory chain.
The study by Brookes and Darley-Usmar (3) shows that the mitochondrial permeability transition (PT) pore is a putative protein complex associated with both the inner and outer mitochondrial membranes. The assembly and opening of this pore is believed to result in swelling and rupture of the outer mitochondrial membrane, leading to the release of cytochrome c and downstream apoptotic signaling. The role of NO in the PT has been controversial and may be concentration dependent, with NO being antiapoptotic at low concentrations and proapoptotic at high concentrations (4). New data are presented indicating that other species, such as Ca2+ and ONOO–, may be potential activators of the PT pore.
Hintze's laboratory (8) presents a study showing that the regulation of oxidative phosphorylation (and cardiac oxygen consumption) by NO depends on the bioavailability of the free radical in the mitochondria. Important determinants are the rates of synthesis and degradation of NO and the diffusion distance between NOS and cytochrome oxidase in the respiratory chain. In the mitochondria, an important indirect regulator of NO degradation is manganese superoxide dismutase (MnSOD or SOD2). This enzyme is a scavenger of superoxide (
), which prevents it from reacting with NO to form ONOO–. Therefore, the higher the levels of MnSOD, the shorter the half-life of and the longer the half-life of NO in the mitochondria. Evidence is presented indicating that NO produced outside of the mitochondria (vascular endothelial cells) is an important regulator of mitochondrial function (9).
Peterson et al. (11) investigates the major route by which excess NO is removed from myocytes, which is widely held to be the stoichiometric reaction of the free radical with oxymyoglobin to form metmyoglobin and nitrate (
) (2, 13). However, evidence is presented suggesting this reaction to be insignificant under most physiological conditions. It is argued that an alternate pathway involving the three-electron reduction of NO to nitrite (
), catalyzed by complex IV (cytochrome oxidase) of the mitochondrial respiratory chain, is the main cellular pathway for NO catabolism (10).
REFERENCES
This article has been cited by other articles:
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  J. Nagendran and E. D. Michelakis Mitochondrial NOS is upregulated in the hypoxic heart: implications for the function of the hypertrophied right ventricle Am J Physiol Heart Circ Physiol, June 1, 2009; 296(6): H1723 - H1726. [Full Text] [PDF]
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 G. C. Brown and V. Borutaite Nitric oxide and mitochondrial respiration in the heart Cardiovasc Res, July 15, 2007; 75(2): 283 - 290. [Abstract] [Full Text] [PDF]
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