A brain mitochondrial nitric oxide synthase: its regulation during rat brain development and its role in mitochondrial H2O2 production

MELANI M; RIOBÓ N; SKVERER S; CARRERAS MC; PODEROSO JJ

Mar del Plata

Congreso; II Congress South American Group for Free Radical Research; 2001

South American Group for Free Radical Research

Nitric oxide has been postulated as a mediator of synaptic plasticity, neuronal differentiation and apoptosis in the central nervous system. In this study, we describe the existence in the rat brain of a NOS exclusively localized in mitochondria (named mtNOS-I), and its developmental regulation. In the adult brain, a protein of 144 kDa was both detected in synaptosomes and mitochondria with antibodies against C-terminal domain of NOS-I, in addition to the classical 157 kDa NOS-I exclusively present in synaptosomes. Neither antibodies against the N-terminal (1-181) region of NOS-I nor against other isoforms recognized the 144 kDa protein. Purified broken mitochondria showed NOS activity with a higher requirement of L-arginine (Km= 12.7 ±1.2 µM) compared with the cytosolic fraction (Km= 2.04 µM). The activity was dependent of Ca2+/CaM, NADPH and BH4 and inhibited by nitro l-arginine. Interestingly during postnatal days 0-4 (P0-P4), only mtNOS-I was detected in the particulate fractions while, from P6 up to adult stage, the 157 kDa isofor was co-expressed with mtNOS-I in this fraction. MtNOS-I was highly active at birth (151 pmol 3H-citrulline min-1mg-1) dropping abruptly between P10-P20 and then recovering a very low activity that persisted in the adult (14 pmol 3H-citrulline min-1mg-1). The contribution of NOS-I was more significant at P10. In addition, MnSOD activity followed a similar temporal pattern that mtNOS-I. In accord, H2O2 production by mitochondria from P0-P4 brains was significantly higher than that from adult brain mitochondria. It is ver interesting the fact that H2O2 production by P0-P4 mitochondria was sharply enhanced by L-arginine addition and suppressed by NO inhibitors. These results suggest the presence of an active NOS-I variant with s distinct N-terminal sequence targeted exclusively to mitohcondris. In conclusion, mitochondrial-derived NO and H2O2 could play a role during rat brain developmental processes.