PODEROSO Juan Jose
Neuronal Nitric Oxide Synthases In Brain And Extraneuronal Tissues.
CARRERAS, MC, MELANI, M, RIOBÓ, N, CONVERSO, DP, GATTO, EM, PODEROSO, JJ
Methods In Enzymology
Año: 2002 vol. 0 p. 1 - 1
Neuronal nitric oxide synthase (nNOSa or NOS I) was the first NOS isoform characterizedand purified in mammalian tissues. 1,2 Since then, nNOS was found in specific neurons ofthe central and peripheral nervous system in a widespread distribution; nNOS is likely toplay an important role in physiologic neuronal functions such as neurotransmitter release,neural development, regeneration, synaptic plasticity and regulation of gene expression butalso, in a variety of neurological disorders in which excessive production of nitric oxide(NO) leads to neural injury.3 In addition, there exist splice isoforms as 136 kDa nNOSb and125 kDa nNOSg.4 In the last years, nNOS isoforms were subsequently reported in nonneuraltissues like rat and human skeletal muscle,5 heart6 and blood circulating cells likeneutrophils.7 Skeletal and cardiac muscle express an alternatively spliced nNOS isoform, a160 kDa nNOSµ which colocalizes with a1-syntrophin and dystrophin at the innermembrane of sarcolemma, near to mitochondria (Table 1).8The subcellular localization of nNOS protein varies greatly among the studied celltypes. In this sense and depending on the study, the particulate enzyme represents between30-60% of the total neuronal nNOS protein; neuronal NOS is a cytosolic enzyme anchoredto postsynaptic PDZ domains and expressed in neurons with different densities, includingthe neocortex, hippocampus, and brainstem.In 1995, Bates et al presented immunocytochemical evidence of the presence ofnitric oxide synthase in non-synaptic brain mitochondria, showing a high percentage ofmitochondria immunolabelled for eNOS at the inner membrane.9 Since then, it has beendescribed the presence of different NOS isoforms in isolated mitochondria of differenttissues. In 1998, Tatoyan et al purified and characterized liver iNOS-like mitochondrialNOS.10 On the other hand, we have recently detected mitochondrial variants related to4nNOS in purified organelles from brain, skeletal muscle and heart; which appears to beslightly different in western blot studies (Fig. 1). In the same way, Kanai et al added strongevidence on the presence of functional neuronal NOS in individual cardiac mitochondria bydirect porphyrinic microsensor measurements.6 These results confirm the existence ofdifferent mtNOS and excludes possible contamination of mitochondrial preparations bycellular isoforms. However, some cross-reactivity of the antibodies against different NOSisoforms could be observed.In brain, mtNOS exhibits a Vmax about 10 times lower than nNOS from wholebrain homogenates (14 ± 3 vs 180 ± 10 pmoles [3H]L-citrulline/min.mg protein,respectively).11 Both, mtNOS (144 kDa) and classical nNOSa (157 kDa) are detected inadult brain synaptosomes with antibodies against C-terminal reductase domain of nNOS(Fig. 2). This 144 kDa protein is not recognized by antibodies against the N-terminal (1-181) region of NOS I or against the other isoforms but eventually, it could react withantibodies detecting longer epitopes in the N-terminal domain, as mentioned by Boveris etal in this volume (see Chapter ).