in vivo 18: 367-376 (2004) Review Regulation of Programmed Cell Death in Neuronal Cells by Nitric Oxide YUN-CHUL KANG1, PETER K. KIM2, BYOUNG-MIN CHOI3, HUN-TAEG CHUNG3, KWON-SOO HA1, YOUNG-GUEN KWON4 and YOUNG-MYEONG KIM1 1Vascular System Research Center and Department of Molecular and Cellular Biochemistry, College of Medicine, Kangwon National University, Chunchon, Kangwon-do, Korea; 2Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, U.S.A; 3Department of Microbiology and Immunology, College of Medicine, Wonkwang University, Iksan, Chonbuk; 4Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Korea Abstract. Nitric oxide (NO), produced from L-arginine and (nNOS), inducible NOS (iNOS) and endothelial NOS molecular oxygen in a reaction catalyzed by one of three NO (eNOS) (1, 2). The functional role of NO can vary by cell synthase isoenzymes, can prevent or induce neuronal apoptosis type and enzyme isotype. The major function of eNOS is depending on its concentration and cellular redox state. This vasodilation by regulating vascular smooth muscle relaxation, molecule affords neuroprotection by post-translational S- while the main role for nNOS may involve neurotransmission nitrosylation of NMDA receptor, caspases and p21ras, and by creating retrograde signaling between synapses. An increases the expression of cytoprotective genes such as HSP70, inducible form of NOS (iNOS) can be up-regulated heme oxygenase and Bcl-2. Moreover, the NO/cGMP pathway considerably by the bacterial cell wall component activates the anti-apoptotic serine/threonine kinase Akt by lipopolysaccharide and/or cytokines which play a role in protein kinase G-dependent activation of phosphatidylinositol regulation of immune cells. At normal intracellular calcium 3-kinase. A high concentration of NO and peroxynitrite, a levels, NO production by iNOS is limited only by the amount reaction product of NO with superoxide anion, can promote of enzyme, substrate, or cofactor present (3, 4) and iNOS apoptotic pathways in neuronal cells through the indirect gene expression at the transcriptional level via the activation activation of caspases. We review the molecular mechanism by of nuclear transcription factor NF-Î B. In contrast, eNOS and which NO exerts both pro- and anti-apoptotic actions in nNOS are constitutively expressed and both are inactive at neuronal cells and the clinical implications for regulating normal calcium concentration. These isoenzymes of NOS neuronal apoptosis. produce puffs of NO in response to transient increases in cytosolic calcium level, but chronic calcium elevation will Function and mode of action of NO in biological system cause persistent NO production (5, 6). Also, the catalytic activity of eNOS can be increased by post-translational Nitric oxide (NO) is a pleiotropic, ubiquitous modulator of modification of palmitoylation and phosphorylation. many cellular functions. NO is synthesized from L-arginine NO interacts directly with several cellular target and molecular oxygen in a reaction catalyzed by one of three molecules, such as thiol, transition metals, molecular oxygen isotypes of NO synthases (NOS) such as neuronal NOS and superoxide, before diffusing out of NO-producing cells. Since NO is small and hydrophobic, it can pass easily through membranes and, as it persists in vivo for a few seconds, NO can diffuse several cell diameters from its site Correspondence to: Young-Myeong Kim, Vascular System of synthesis (7). When immune-activated hepatocytes were Research Center and Department of Molecular and Cellular co-cultured with red blood cells, the biological scavengers of Biochemistry, College of Medicine, Kangwon National University, Chunchon, Kangwon-Do 200-701, Republic of Korea. Tel: +82-33- NO, the nitrosyl iron complexes were mostly formed in red 250-8831, Fax: +82-33-244-3286, e-mail: [email protected] blood cells, but few within NO-producing hepatocytes (8). This result indicates that the transcellular diffusion of NO is Key Words: Nitric oxide, neuronal cells, apoptosis, review. more rapid than the rate of its intracellular reaction in NO- 0258-851X/2004 $2.00+.40 367 in vivo 18: 367-376 (2004) generating cells. Consequently the steady-state NO formation of dinitrosyl iron complexes may be involved in concentration experienced by a cell is determined by the transnitrosylation to the redox-sensitive sulfhydryl group of number of NO-producing cells nearby (7). NO produced several biomolecules responsible for apoptotic cell death, within cells, therefore, interacts with biomolecules through resulting in the modulation of neuronal apoptotic cell death. both autocrine and paracrine action modes. Types of cell death Biochemical reactivity with biological molecules Programmed cell death (PCD) is a normal physiological NO can interact both with oxygen and a great number of other process that occurs during embryonic development as well as in biological molecules such as transition metals, redox-sensitive the maintenance of tissue homeostasis. Eukaryotic cells that thiol and tyrosine residues. Although NO reacts with O2 in die by PCD undergo an astonishingly stereotypical series of aqueous solution to produce nitrite, this reaction is very slow at biochemical and morphological changes. PCD is believed to physiological concentrations of O2. NO reacts rapidly with occur by caspase-dependent and -independent mechanisms - superoxide anion (O2 ) and forms the strong oxidant (17, 18). These two mechanisms have distinct histological and peroxynitrite (ONOO-), a cytotoxic modulator in neuronal cells. biochemical signatures. Caspase-dependent PCD, or apoptosis, NO also interacts with many iron-containing proteins, such as involves the activation of a cascade of events that orchestrate hemoproteins and nonheme-iron-containing proteins. NO the destruction of the cell and thus apoptosis is blocked by reacts with the heme iron of hemoglobin and myoglobin to inhibition of caspase activation and activity. Apoptosis is a form nitrate. NO binds the heme moiety of soluble guanylate strictly regulated device responsible for the ordered removal cyclase and increases its catalytic activity to generate more of superfluous, aged and damaged cells (19, 20). cGMP from GTP. The augmentation of this enzymatic activity Morphologically, in cells undergoing apoptosis there is ruffling, plays a key role in regulation of vascular tone and blebbing and condensation of the plasma and nuclear neurotransmission (9). Binding of NO to heme or the iron- membranes and, subsequently, aggregation of nuclear sulfur cluster of enzymes inhibits their catalytic activity such as chromatin. Mitochondria and ribosomes retain their gross cytochrome oxidase, catalase, cytochrome P-450, ferrochelatase structure and at least partial function. There is disruption of and aconitase (10). The reaction of NO with biological the cytoskeletal architecture; the cell shrinks and then - components such as metals, thiols, O2 and O2 produces a fragments into a cluster of membrane-enclosed "apoptotic variety of secondary products ranging from the innocuous bodies" that are rapidly ingested by adjacent macrophages or - - oxidized components (NO2 and NO3 ) to the reactive nitrogen other neighboring phagocytic cells. Apoptotic cells display a intermediates such as nitrosonium equivalent (NO+), ONOO, characteristic pattern of DNA involving fragmentation into - S-nitrosothiol, nitroxyl ion (NO ), dinitrogen trioxide (N2O3) distinct segments that can be visualized as a ladder of bands by and nitrogen dioxide (NO2). These reactive nitrogen species, gel electrophoresis. Other features of apoptosis are a reduction including NO, interact with other biomolecules to cause lipid in the membrane potential of mitochondria, intracellular oxidation, protein modification (e.g. cysteine and tyrosine acidification, generation of free radicals and externalization of residue), DNA damage or base modification and regulation of phosphatidylserine residues (21, 22). enzyme activity. Contrary to earlier expectations, the inhibition of caspase Because NO is a radical molecule and does not effectively activation does not necessarily protect against cell death nitrosylate thiol groups, a NO reaction product is implicated stimuli. Instead it might reveal, or even enhance, underlying in the post-translational S-nitrosylation of cysteine residue. caspase-independent cell death programs. Although these NO+ equivalent can nitrosylate thiols (11). Free endogenous programs might resemble apoptosis-like PCD, the caspase- NO+ exists only at low pH, such as that of the stomach (12). independent mode includes two patterns of cell death, such N2O3 formed by the reaction of NO with molecular oxygen as "apoptosis-like PCD" and "necrosis-like PCD". In has a strong propensity to nitrosate both amine and thiol apoptosis-like PCD, chromatin may condense, but not to the moieties at physiological pH (12). Reactive nitrogen oxide characteristic geometric shape associated with classic species, including NO+ and its equivalents, can be generated apoptosis, and phagocytosis-recognition molecules including by the reaction of NO with O2 (13), iron-sulfur clusters (13), phosphatidylserine are displayed before cell lysis of the or heme iron (15). We have demonstrated that chemically plasma membrane. In necrosis-like PCD, chromatin may not synthesized dinitrosyl iron complexes induce post- condense at all, but other apoptosis-like features, including translational S-nitrosylation