A quantitative assessment of the levels of cytochromes P-450 b and P-450 c in the brains and pituitary glands of untreated and β-naphthoflavone (BNF)-pretreated rats was made with polyclonal antibodies raised against hepatic P-450 b and c and the senstive fluorometric assay of P-450 catalytic activity, namely, the O-deethylation of ethoxycoumarin (ETC). In the microsomal fraction of brains of untreated rats, the rate of formation of 7-hydrodxycoumarin from ETC ranged between 0.1 and 20 pmol/min/mg of microsomal protein, which is approximately 0.01-2% of the level of hepatic microsomes of phenobarbital-induced rats. This brain activity was completely inhibited by anti P-450 b antibodies but was unaffected by anti P-450 c antibodies. As with hepatic P-450 b, metyrapone and chloramphenicol (100 μM) were good inhibitors of catalytic activity, whereas α-naphthoflavone (1 μM) was a poor inhibitor. No ETC O-deethylase activity was detectable in microsomes prepared from the pituitary glands of untreated rats. Upon pretreatment of rats with BNF, there was induction of ETC O-deethylase activity in the pituitary gland to a level of 3.3±1.5 pmol/min/mg of microsomal protein, but there was no significant increase in the level of activity in brain microsomes. Despite this, there was evidence of induction of P-450 c in both the brain and pituitary of BNF-pretreated rats since anti P-450 c antibodies inhibited brain activity by 55% and pituitary activity by 84%. The regional distribution of P-450 b and c in the hypothalamic-preoptic area and olfactory bulbs was examined. The level of ETC O-deethylase activity in the hypothalamic-preoptic area was not different from that in the whole brain, but in the olfactory bulbs activity was higher than that in whole brain, with a range of 0.1-52 pmol/min/mg of microsomal protein. The catalytic activity in the whole brain and in the olfactory bulbs was inhibited by anti P-450(b) but not by anti P-450(c) antibodies. Neither estradiol, testosterone, dehydroterstosterone, nor 5α-androstane,3β,17β-diol (100 μM) competitively inhibited ETC O-deethylase activity, indicating that P-450 b is not responsible for the steroid hydroxylations previously reported in the brain. BNS pretreatment of rats did not cause a consistent increase in ETC O-deethylase upon BNF induction. However, there was an induction of P-450 c in the olfactory bulbs since catalytic activity was inhibited with anti P-450(c) antibodies. Western immunoblots with microsomes from whole brain, hypothalamic-preoptic area, or olfactory bulbs of control or BNF-pretreated rats gave no signals with either P-450 b or P-450 c antibodies. This is perhaps not surprising, since the level of these enzymes is less than 0.1% of that in liver microsomes, which means that the P-450 proteins constitute approximately 20 ng/mg of microsomal protein and this is below the level of detectability of the Western immunoblots.
|Original language||English (US)|
|Number of pages||7|
|State||Published - Jan 1 1988|
ASJC Scopus subject areas
- Molecular Medicine