61ª Reunião Anual da SBPC
C. Ciências Biológicas - 3. Bioquímica - 4. Metabolismo e Bioenergética
ROLE  OF NITRIC OXIDE  INDUCING MEMBRANE PERMEABILITY TRANSITION IN RAT LIVER MITOCHONDRIA
Rafael Garcia 1
Fabiane Leonel Utino 1
Ana Catarina Rezende Leite 1, 2
Roger Frgério Castilho 1
Helena Coutinho Franco Oliveira 2
Aníbal Eugênio Vercesi 1
1. Departamento de Patologia Clínica, Faculdade de Ciências Médicas
2. Departamento de Fisiologia e Biofísica, Instituto de Biologia
INTRODUÇÃO:

Nitric oxide (NO·) generated in mitochondria seems to regulate energy metabolism, O2 consumption, and reactive oxygen species (ROS) formation by this organelle. The synthesis of NO· from L-arginine and O2 is catalyzed, in mitochondria, by a calcium-dependent nitric oxide synthase (mtNOS). Therefore, the activity of this mtNOS is linked to the critical function of mitochondria in maintaining matrix calcium homeostasis. In this regard, mitochondrial permeability transition (MPT), a non-selective permeabilization of the inner mitochondrial membrane is triggered by the accumulation of excessive quantities of Ca2+ in mitochondria. High concentrations of NO· and its derivatives can promote MPT, whereas low rates of NO· formation can inhibit Ca2+ accumulation and thus prevent MPT. Previous results from our group have shown that NOS inhibitors induce Ca2+-dependent MPT in rat liver mitochondria.

METODOLOGIA:

Mitochondrial swelling was determined as the decrease in the turbidity of the mitochondrial suspension measured in a spectrophotometer. Mitochondrial membrane potencial disruption - Safranin O fluorescence was used to estimate mitochondrial membrane potentials by following its fluorescence on a spectrofluorometer. Mitochondrial calcium uptake - Calcium uptake by isolated liver mitochondria was monitored following the fluorescence of calcium green-5N hexapotassium salt.  Mitochondrial nitric oxide production – DAF-FM was used to monitor RNS release rates in rat liver mitochondrial suspension. RNS production was measured using a spectrofluorometer.

RESULTADOS:

In the present study we show that low amounts of SNAP (a NO· donor) can avoid MPT triggered by Ca2+ and protect against MPT stimulated by L-NAME (a NOS inhibitor). It was also observed that SNAP, at low quantities, not only delays Ca2+ release from control mitochondria, but also from those treated with L-NAME. A direct effect of L-NAME on MPT was ruled out by the lack of effect of its isomer D-NAME.

CONCLUSÃO:

In the light of these results, we conclude that the release of physiological amounts of NO· by NO· donors can partially inhibit MPT brought about by the inhibition of the mtNOS activity.

Instituição de Fomento: CAPES, CNPq and FAPESP
Palavras-chave: Mitochondrial Permeability Transition, Nitric Oxide, SNAP.