Alterations in the expression of Nitric Oxide Synthase due to porphyrinogenic drugs

SAMPAYO, ROCIO; LAVANDERA, JIMENA; BATLLE, ALCIRA; BUZALEH, ANA MARIA

Stockholm

Congreso; PORPHYRINS & PORPHYRIAS; 2009

The existence of heme as a functional prosthetic group in a large variety of housekeeping proteins is crucial for life and death. Under heme deficiency conditions, cells fail to survive and subsequently die because of impairment of major vital functions carried out by hemeproteins. Porphyrias neuropathophysiology could be related to low levels of heme, a cofactor for nitric oxide synthase (NOS), enzyme responsible of NO synthesis in mammalian tissues. Four NOS isoforms have been described: neuronal NOS (nNOS), endothelial NOS (eNOS), inducible NOS (iNOS) and mitochondrial NOS (mtNOS). To provide further understanding on the onset of the porphyrias, we examined how anaesthetics and other porphyrinogenic agents affect nitric oxide metabolism in brain and liver of mice. A comparative study in animals receiving 5-aminolevulinic acid (ALA) was carried out. Recently, we have reported that NOS activity response depends on both the porphyrinogenic agent and the cellular fraction analyzed. Ethanol, chronic enflurane and isoflurane increased liver cytosolic and brain mitochondrial activity; while oral griseofulvin enhanced liver mitochondrial activity. ALA also produced a diminution of NOS activity in both tissues. In this work, the expression of nNOS, mtNOS and iNOS was measured in brain. Western blot analysis identified a protein of 157 kDa reacting with anti-nNOS antibodies (amino terminus) in cytosolic fraction and other protein of 144 kDa reacting with anti-nNOS antibodies in mitochondrial fraction (mtNOS). nNOS expression was diminished after acute (90%) and chronic (43%) enflurane, veronal (60%) and ethanol (87%) and enhanced after acute isoflurane (40%) (p<0.05, n=4). mtNOS expression was induced after chronic enflurane (45%) and diminished after ethanol (50%), topical Gris (80%) and starvation (75%) (p<0.05, n=4). No induction of iNOS was detected in any of the assayed treatments. In liver, NOS expression also varied in function of the agent studied. ALA affected liver nNOS expression. NOS expression diminution could be a consequence of free heme pool reduction. Results would also indicate the development of nitrosative stress besides the oxidative stress previously demonstrated and give evidence about the widespread action of porphyrinogenic drugs in brain.