Rat brain cytochromes P-450: catalytic, immunochemical properties and inducibility of multiple forms

Cytochrome P-450 (P-450) and associated mono-oxygenase activities were estimated in male and female rat brain microsomes. The P-450 concentration in male rat brain was one-tenth the corresponding hepatic levels, which is considerably higher than earlier reports. A distinct sex-related difference was observed in the levels of total P-450 and mono-oxygenase activities known to be mediated by P-450b,e; the female brain levels were 60% of those in the males. Immunoinhibition and immunoblot studies using antisera to P-450b,e and P-450c,d indicated the presence of multiple forms of P-450, immunologically similar to P-450b,e, P-450c and P-450d in the rat brain. Prior treatment with phenobarbital resulted in two-fold increase of total P-450 and selective induction of aminopyrine N-demethylase (APD) and morphine N-demethylase (MND) activities. Administration of 3-methylcholanthrene, selectively induced the levels of ethoxycoumarin O-deethylase (ECD) and arylhydrocarbon hydroxylase, although the levels of total P-450 were not increased. 3-Methylcholanthrene induction was also accompanied by a shift in the absorption maximum of the reduced carbon monoxide difference spectrum from 452 to 448 nm. Immunocytochemical localization using antibodies to P-450b,e indicated the presence of P-450 predominantly in the neuronal cell bodies and to a lesser extent in the fibre tracts in cerebral cortex, cerebellum, thalamus, hypothalamus, hippocampus and brainstem. These studies indicate that the brain contains significant amounts of P-450, which exists in multiple forms and can be selectively induced by prior exposure to phenobarbital or 3-methylcholanthrene

Low glutathione levels in brain regions of aged rats

Glutathione (GSH) was measured in 6 regions of brain and liver of young adult, middle-aged and aged rats. GSH levels were significantly lower in cortex, cerebellum, striatum, thalamus and hippocampus of aged rats, while no changes were observed in liver as compared to young adult rats. On the other hand, lipid peroxidation as measured by thiobarbituric acid-reactive products increased significantly in all the regions of brain examined and in the liver of aged rats. Since GSH plays an important role as a cellular protectant against oxygen radical-mediated injury, decreased levels of GSH in aged rat brain are indicative of the vulnerability of the aged cerebral tissue to oxidative injury

Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells

Alzheimer's amyloid precursor protein 695 (APP) is a plasma membrane protein, which is known to be the source of the toxic amyloid β (Aβ) peptide associated with the pathogenesis of Alzheimer's disease (AD). Here we demonstrate that by virtue of its chimeric NH2-terminal signal, APP is also targeted to mitochondria of cortical neuronal cells and select regions of the brain of a transgenic mouse model for AD. The positively charged residues at 40, 44, and 51 of APP are critical components of the mitochondrial-targeting signal. Chemical cross-linking together with immunoelectron microscopy show that the mitochondrial APP exists in NH2-terminal inside transmembrane orientation and in contact with mitochondrial translocase proteins. Mutational studies show that the acidic domain, which spans sequence 220–290 of APP, causes the transmembrane arrest with the COOH-terminal 73-kD portion of the protein facing the cytoplasmic side. Accumulation of full-length APP in the mitochondrial compartment in a transmembrane-arrested form, but not lacking the acidic domain, caused mitochondrial dysfunction and impaired energy metabolism. These results show, for the first time, that APP is targeted to neuronal mitochondria under some physiological and pathological conditions

Mitochondrial trafficking of APP and alpha synuclein: Relevance to mitochondrial dysfunction in Alzheimer's and Parkinson's diseases

AbstractMitochondrial dysfunction is an important intracellular lesion associated with a wide variety of diseases including neurodegenerative disorders. In addition to aging, oxidative stress and mitochondrial DNA mutations, recent studies have implicated a role for the mitochondrial accumulation of proteins such as plasma membrane associated amyloid precursor protein (APP) and cytosolic alpha synuclein in the pathogenesis of mitochondrial dysfunction in Alzheimer's disease (AD) and Parkinson's disease (PD), respectively. Both of these proteins contain cryptic mitochondrial targeting signals, which drive their transport across mitochondria. In general, mitochondrial entry of nuclear coded proteins is assisted by import receptors situated in both outer and inner mitochondrial membranes. A growing number of evidence suggests that APP and alpha synclein interact with import receptors to gain entry into mitochondrial compartment. Additionally, carboxy terminal cleaved product of APP, ∼4 kDa Abeta, is also transported into mitochondria with the help of mitochondrial outer membrane import receptors. This review focuses on the mitochondrial targeting and accumulation of these two structurally different proteins and the mode of mechanism by which they affect the physiological functions of mitochondria

Administration of testosterone alleviates the constitutive sex difference in rat brain cytochrome P-450

Sex-related difference was observed in the levels of total cytochrome P-450 (P-450) and the mono-oxygenase activity mediated by P-450(b,e), namely, aminopyrine N-demethylase and morphine N-demethylase activity in rat brain microsomes. Male rat brain had higher activity of the above enzymes as compared to the female rat brain. On the other hand, P-450(c,d) mediated 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase activity showed no sex-related difference in rat brain. Administration of testosterone elevated the levels of total P-450, aminopyrine N-demethylase and morphine N-demethylase in female rat brain to levels comparable with that of the male rat brain. No significant change was observed in the levels of 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase and NADPH cytochrome c reductase. All of the above enzyme levels were unaffected in the male rat brain following the treatment with testosterone. These results indicate that testosterone may regulate the forms of cerebral P-450 that are associated with the sex-related difference observed in rat brain

Preparation of brain microsomes with cytochrome P450 activity using calcium aggregation method

Microsomes have been conventionally prepared by centrifugation of the postmitochondrial supernatant at 100,000g using an ultracentrifuge. Liver microsomes have been prepared by low speed centrifugation following sedimentation of the microsomal membranes in the presence of calcium ions. However, this method has not been suitable for the preparation of microsomes from extrahepatic tissues as it often results in the loss of cytochrome P450 activity. Brain microsomes prepared by the traditional calcium aggregation method results in the loss of cytochrome P450. We now describe a modification of the calcium aggregation method for the rapid preparation of rat and mouse brain microsomes. This involves the incorporation of glycerol, dithiothreitol, and EDTA in the preparation of microsomes. Such preparations do not differ in their cytochrome P450 content and associated monooxygenase activity from the traditionally prepared microsomes using ultracentrifugation. Electron microscopic analysis also does not reveal any differences between the microsomes prepared by the two methods. As brain microsomes are relatively unstable and are obtained in low yields, rapid isolation of large quantities of microsomes, possible using the present method, should be very useful

NADPH cytochrome P-450 reductase in rat, mouse and human brain

NADPH cytochrome P-450 reductase (P-450 reductase), an essential component of the cytochrome P-450 mono-oxygenase system, has been estimated in rat and mouse brain, and seven human brains obtained at autopsy. The ratio of cytochrome P-450 to P-450 reductase is lower in the rat and mouse brains (2.5-4.0) as compared to the respective livers (10.0-11.0). The rat and mouse brain P-450 reductase were immunologically similar to the rat liver P-450 reductase as examined by immunochemical inhibition, Ouchterlony double diffusion and immunoblot. The antisera to rat liver P-450 reductase inhibited rat brain aminopyrine N-demethylase activity to the same extent as NADPH cytochrome c reductase, suggesting that the level of P-450 reductase controls the rate of this cytochrome P-450 mediated activity. The human brain NADPH cytochrome c reductase exhibited regional variation, maximal activity being observed in the brain stem region. Immunochemical inhibition and immunoblot studies revealed immunological cross-reactivity between rat liver reductase and human brain medulla, while none was observed in cortex or cerebellum. Immunocytochernical studies on human brain medulla using antisera to rat liver P-450 reductase indicated localization of the P-450 reductase in neuronal cell body