Mechanism of mitochondrial stress-induced resistance to apoptosis in mitochondrial DNA-depleted C2C12 myocytes.

In this study, we show that partial mitochondrial DNA (mtDNA) depletion (mitochondrial stress) induces resistance to staurosporine (STP)-mediated apoptosis in C2C12 myoblasts. MtDNA-depleted cells show a 3-4-fold increased proapoptotic proteins (Bax, BAD and Bid), markedly increased antiapoptotic Bcl-2, and reduced processing of p21 Bid to active tBid. The protein levels and also the ability to undergo STP-mediated apoptosis were restored in reverted cells containing near-normal mtDNA levels and restored mitochondrial transmembrane potential. Inhibition of apoptosis closely correlated with sequestration of Bax, Bid and BAD in the mitochondrial inner membrane, increased Bcl-2 and Bcl-X(L), and inability to process p21 Bid. These factors, together with the reduced activation of caspases 3, 9 and 8 are possible causes of mitochondrial stress-induced resistance to apoptosis. Our results suggest that a highly proliferative and invasive behavior of mtDNA-depleted C2C12 cells is related to their resistance to apoptosis.

Evolutionarily divergent electron donor proteins interact with P450MT2 through the same helical domain but different contact points.

We have investigated the sites of N-terminally truncated cytochrome P4501A1 targeted to mitochondria (P450MT2) which interact with adrenodoxin (Adx), cytochrome P450 reductase (CPR) and bacterial flavodoxin (Fln). The binding site was mapped by a combination of in vitro mutagenesis, in vivo screening with a mammalian two-hybrid system, spectral analysis, reconstitution of enzyme activity and homology-based structural modeling. Our results show that part of an aqueous accessible helix (putative helix G, residues 264-279) interacts with all three electron donor proteins. Mutational studies revealed that Lys267 and Lys271 are crucial for binding to Adx, while Lys268 and Arg275 are important for binding to CPR and FLN: Additive effects of different electron donor proteins on enzyme activity and models on protein docking show that Adx and CPR bind in a non-overlapping manner to the same helical domain in P450MT2 at different angular orientations, while CPR and Fln compete for the same binding site. We demonstrate that evolutionarily divergent electron donor proteins interact with the same domain but subtly different contact points of P450MT2.

Mitochondrial targeted cytochrome P450 2E1 (P450 MT5) contains an intact N terminus and requires mitochondrial specific electron transfer proteins for activity.

Hepatic mitochondria contain an inducible cytochrome P450, referred to as P450 MT5, which cross-reacts with antibodies to microsomal cytochrome P450 2E1. In the present study, we purified, partially sequenced, and determined enzymatic properties of the rat liver mitochondrial form. The mitochondrial cytochrome P450 2E1 was purified from pyrazole-induced rat livers using a combination of hydrophobic and ion-exchange chromatography. Mass spectrometry analysis of tryptic fragments of the purified protein further ascertained its identity. N-terminal sequencing of the purified protein showed that its N terminus is identical to that of the microsomal cytochrome P450 2E1. In reconstitution experiments, the mitochondrial cytochrome P450 2E1 displayed the same catalytic activity as the microsomal counterpart, although the activity of the mitochondrial enzyme was supported exclusively by adrenodoxin and adrenodoxin reductase. Mass spectrometry analysis of tryptic fragments and also immunoblot analysis of proteins with anti-serine phosphate antibody demonstrated that the mitochondrial cytochrome P450 2E1 is phosphorylated at a higher level compared with the microsomal counterpart. A different conformational state of the mitochondrial targeted cytochrome P450 2E1 (P450 MT5) is likely to be responsible for its observed preference for adrenodoxin and adrenodoxin reductase electron transfer proteins.

Physical interaction and functional synergy between glucocorticoid receptor and Ets2 proteins for transcription activation of the rat cytochrome P-450c27 promoter.

We demonstrate that dexamethasone-mediated transcription activation of the cytochrome P-450c27 promoter involves a physical interaction and functional synergy between glucocorticoid receptor (GR) and Ets2 factor. Ets2 protein binding to a "weak" Ets-like site of the promoter is dependent on GR bound to the adjacent cryptic glucocorticoid response element. Coimmunoprecipitation and chemical cross-linking experiments show physical interaction between GR and Ets2 proteins. Mutational analyses show synergistic effects of Ets2 and GR in dexamethasone-mediated activation of the cytochrome P-450c27 promoter. The DNA-binding domain of GR, lacking the transcription activation and ligand-binding domains, was fully active in synergistic activation of the promoter with intact Ets2. The DNA-binding domain of Ets2 lacking the transcription activation domain showed a dominant negative effect on the transcription activity. Finally, a fusion protein consisting of the GR DNA-binding domain and the transcription activation domain of Ets2 fully supported the transcription activity, suggesting a novel synergy between the two proteins, which does not require the transactivation domain of GR. Our results also provide new insights on the role of putative weak consensus Ets sites in transcription activation, possibly through synergistic interaction with other gene-specific transcription activators.

Accumulation of mitochondrial P450MT2, NH(2)-terminal truncated cytochrome P4501A1 in rat brain during chronic treatment with beta-naphthoflavone. A role in the metabolism of neuroactive drugs.

The biochemical and molecular characteristics of cytochrome P4501A1 targeted to rat brain mitochondria was studied to determine the generality of the targeting mechanism previously described for mitochondrial cytochrome P450MT2 (P450MT2) from rat liver. In rat brain and C6 glioma cells chronically exposed to beta-naphoflavone (BNF), P450MT2 content reached 50 and 95% of the total cellular pool, respectively. P450MT2 from 10 days of BNF-treated rat brain was purified to over 85% purity using hydrophobic chromatography followed by adrenodoxin affinity binding. Purified brain P450MT2 consisted of two distinct molecular species with NH(2) termini identical to liver mitochondrial forms. These results confirm the specificity of endoprotease-processing sites. The purified P450MT2 showed a preference for adrenodoxin + adrenodoxin reductase electron donor system and exhibited high erythromycin N-demethylation activity. Brain mitoplasts from 10-day BNF-treated rats and also purified P450MT2 exhibited high N-demethylation activities for a number of neuroactive drugs, including trycyclic anti-depressants, anti-convulsants, and opiates. At 10 days of BNF treatment, the mitochondrial metabolism of these neuroactive drugs represented about 85% of the total tissue activity. These results provide new insights on the role of P450MT2 in modulating the pharmacological potencies of different neuroactive drugs in chronically exposed individuals.

Novel biochemical and functional insights into nuclear Ca(2+) transport through IP(3)Rs and RyRs in osteoblasts.

We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab(34)) and anti-IP(3)R (Ab(40)) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific anti-receptor antisera, notably Ab(40), anti-RyR-1, anti-RyR-2 (Ab(129)), and anti-RyR-3 (Ab(180)). Only anti-RyR-1 and Ab(40) showed bands corresponding, respectively, to full-length RyR-1 ( approximately 500 kDa) and IP(3)R-1 (approximately 250 kDa). Band intensity was reduced by just approximately 20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca(2+) concentration ([Ca(2+)](np)) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca(2+) via Ca(2+)-ATPase activation (1 mM ATP and approximately 100 nM Ca(2+)). Adequate Ca(2+) loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca(2+)-loaded nuclei to IP(3) or cADP ribose resulted in a rapid and sustained [Ca(2+)](np) elevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca(2+) influx in osteoblasts through nuclear membrane-resident IP(3)Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP(3)-Ca(2+) pathway.

A new function for CD38/ADP-ribosyl cyclase in nuclear Ca2+ homeostasis.

Nucleoplasmic calcium ions (Ca2+) influence nuclear functions as critical as gene transcription, apoptosis, DNA repair, topoisomerase activation and polymerase unfolding. Although both inositol trisphosphate receptors and ryanodine receptors, types of Ca2+ channel, are present in the nuclear membrane, their role in the homeostasis of nuclear Ca2+ remains unclear. Here we report the existence in the inner nuclear membrane of a functionally active CD38/ADP-ribosyl cyclase that has its catalytic site within the nucleoplasm. We propose that the enzyme catalyses the intranuclear cyclization of nicotinamide adenine dinucleotide to cyclic adenosine diphosphate ribose. The latter activates ryanodine receptors of the inner nuclear membrane to trigger nucleoplasmic Ca2+ release.

Dual targeting of cytochrome P4502B1 to endoplasmic reticulum and mitochondria involves a novel signal activation by cyclic AMP-dependent phosphorylation at ser128.

We have investigated mechanisms of mitochondrial targeting of the phenobarbital-inducible hepatic mitochondrial P450MT4, which cross-reacts with antibody to microsomal P4502B1. Results show that P4502B1 and P450MT4 have identical primary sequence but different levels of phosphorylation and secondary structure. We demonstrate that P4502B1 contains a chimeric mitochondrial and endoplasmic reticulum (ER) targeting signal at its N-terminus. Inducers of cAMP and protein kinase A-mediated phosphorylation of P4502B1 at Ser128 activate the signal for mitochondrial targeting and modulate its mitochondrial or ER destination. S128A mutation inhibits in vitro mitochondrial transport and also in vivo mitochondrial targeting in COS cells. A fragment of P4502B1 containing the N-terminal signal and the phosphorylation site could drive the transport of dihydrofolate reductase (DHFR) into mitochondria. Ser128 phosphorylation reduced the affinity of 2B1 protein for binding to SRP, but increased the affinity of the 2B1-DHFR fusion protein for binding to yeast mitochondrial translocase proteins, TOM40 and TIM44, and matrix Hsp70. We describe a novel regulatory mechanism by which cAMP modulates the targeting of a protein to two distinct organelles.

CD38/ADP-ribosyl cyclase: A new role in the regulation of osteoclastic bone resorption.

The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.

Dual targeting property of the N-terminal signal sequence of P4501A1. Targeting of heterologous proteins to endoplasmic reticulum and mitochondria.

Recent studies from our laboratory showed that the beta-naphthoflavone-inducible cytochrome P4501A1 is targeted to both the endoplasmic reticulum (ER) and mitochondria. In the present study, we have further investigated the ability of the N-terminal signal sequence (residues 1-44) of P4501A1 to target heterologous proteins, dihydrofolate reductase, and the mature portion of the rat P450c27 to the two subcellular compartments. In vitro transport and in vivo expression experiments show that N-terminally fused 1-44 signal sequence of P4501A1 targets heterologous proteins to both the ER and mitochondria, whereas the 33-44 sequence strictly functions as a mitochondrial targeting signal. Site-specific mutations show that positively charged residues at the 34th and 39th positions are critical for mitochondrial targeting. Cholesterol 27-hydroxylase activity of the ER-associated 1-44/1A1-CYP27 fusion protein can be reconstituted with cytochrome P450 reductase, but the mitochondrial associated fusion protein is functional with adrenodoxin + adrenodoxin reductase. Consistent with these differences, the fusion protein in the two organelle compartments exhibited distinctly different membrane topology. The results on the chimeric nature of the N-terminal signal of P4501A1 coupled with interaction with different electron transport proteins suggest a co-evolutionary nature of some of the xenobiotic inducible microsomal and mitochondrial P450s.

Retrograde Ca2+ signaling in C2C12 skeletal myocytes in response to mitochondrial genetic and metabolic stress: a novel mode of inter-organelle crosstalk.

We have investigated the mechanism of mitochondrial-nuclear crosstalk during cellular stress in mouse C2C12 myocytes. For this purpose, we used cells with reduced mitochondrial DNA (mtDNA) contents by ethidium bromide treatment or myocytes treated with known mitochondrial metabolic inhibitors, including carbonyl cyanide m-chlorophenylhydrazone (CCCP), antimycin, valinomycin and azide. Both genetic and metabolic stresses similarly affected mitochondrial membrane potential (Deltapsim) and electron transport-coupled ATP synthesis, which was also accompanied by an elevated steady-state cytosolic Ca2+ level ([Ca2+]i). The mitochondrial stress resulted in: (i) an enhanced expression of the sarcoplasmic reticular ryanodine receptor-1 (RyR-1), hence potentiating the Ca2+ release in response to its modulator, caffeine; (ii) enhanced levels of Ca2+-responsive factors calineurin, calcineurin-dependent NFATc (cytosolic counterpart of activated T-cell-specific nuclear factor) and c-Jun N-terminal kinase (JNK)-dependent ATF2 (activated transcription factor 2); (iii) reduced levels of transcription factor, NF-kappaB; and (iv) enhanced transcription of cytochrome oxidase Vb (COX Vb) subunit gene. These cellular changes, including the steady-state [Ca2+]i were normalized in genetically reverted cells which contain near-normal mtDNA levels. We propose that the mitochondria-to-nucleus stress signaling occurs through cytosolic [Ca2+]i changes, which are likely to be due to reduced ATP and Ca2+ efflux. Our results indicate that the mitochondrial stress signal affects a variety of cellular processes, in addition to mitochondrial membrane biogenesis.

Physiological role of the N-terminal processed P4501A1 targeted to mitochondria in erythromycin metabolism and reversal of erythromycin-mediated inhibition of mitochondrial protein synthesis.

Recently, we showed that the major species of beta-naphthoflavone-inducible rat liver mitochondrial P450MT2 consists of N-terminal truncated microsomal P4501A1 (+33/1A1) and that the truncated enzyme exhibits different substrate specificity as compared with intact P4501A1. The results of the present study show that P450MT2 targeted to COS cell mitochondria by transient transfection of P4501A1 cDNA is localized inside the mitochondrial inner membrane in a membrane-extrinsic orientation. Co-expression with wild type P4501A1 and adrenodoxin (Adx) cDNAs resulted in 5-7-fold higher erythromycin N-demethylation (ERND) in the mitochondrial fraction but minimal changes in the microsomal fraction of transfected cells. Erythromycin, a potent inhibitor of bacterial and mitochondrial protein synthesis, caused 8-12-fold higher accumulation of CYP1A1 mRNA, preferential accumulation of P450MT2, and 5-6-fold higher ERND activity in the mitochondrial compartment of rat C6 glioma cells. Consistent with the increased mitochondrial ERND activity, co-expression with P4501A1 and Adx in COS cells rendered complete protection against erythromycin-mediated mitochondrial translation inhibition. Mutations that specifically affect the mitochondrial targeting of P4501A1 also abolished protection against mitochondrial translation inhibition. These results for the first time suggest a physiological function for the xenobiotic inducible cytochrome P4501A1 against drug-mediated mitochondrial toxicity.

Interaction of adrenodoxin with P4501A1 and its truncated form P450MT2 through different domains: differential modulation of enzyme activities.

Recently we showed that the beta-naphthoflavone-inducible liver mitochondrial P450MT2 consists of two N-terminal truncated forms of the microsomal P4501A1, termed P450MT2a (+5/1A1) and MT2b (+33/1A1) [Addya et al. (1997) J. Cell Biol. 139, 589-599]. In the present study, we demonstrate that intact P4501A1 and the major mitochondrial form, P450MT2b (routinely referred to as P450MT2), show distinct substrate specificities and preference for different electron transport proteins. Enzyme reconstitution and spectral studies show that the wild-type adrenodoxin (Adx), but not the mutant Adx, binds to P450MT2 in a functionally productive manner (Kd = 0.6 microM) and induces a characteristic high-spin state. Adx binding to intact P4501A1 or +5/1A1 is less efficient as seen from spectral shift patterns (Kd = 1.8-2.0 microM) and reconstitution of enzyme activity. Use of Adx--Sepharose affinity matrix yielded < 90% pure P450MT2 (specific activity: 13.5 nmol/mg of protein) starting from a partially purified fraction of 10-15% purity, further demonstrating the specificity of P450MT2 and Adx interaction. Chemical cross-linking studies show that the bovine Adx forms heteroduplexes with both P450MT2 and intact P4501A1, though at different efficiencies. Our results show that Adx interacts with P450MT2 through its C-terminal acidic domain 2, while interaction with intact P4501A1 likely involves the N-terminal acidic domain 1. These results point to an interesting possibility that different electron transfer proteins may differently modulate the enzyme activity. Our results also demonstrate for the first time as to how a different mode of Adx interaction differently modulates the substrate specificities of the two P450 forms.

Targeting of NH2-terminal-processed microsomal protein to mitochondria: a novel pathway for the biogenesis of hepatic mitochondrial P450MT2.

Cytochrome P4501A1 is a hepatic, microsomal membrane-bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from beta-naphthoflavone-induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30-amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33-44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.

Localization of multiple forms of inducible cytochromes P450 in rat liver mitochondria: immunological characteristics and patterns of xenobiotic substrate metabolism.

Hepatic mitochondria contain inducible cytochromes P450 that cross-react with antibodies to P4501A1/2 and 2B1/2. In the present study, we present evidence for the occurrence of additional P450 forms in rat liver mitochondria that cross-react with antibodies to microsomal P4503A1/2 and 2E1. Protease protection and also immunoelectron microscopy studies were carried out to support the mitochondrial location of the immunoreactive P450s. The solubility of immunoreactive proteins in 0.1 M Na2CO3 suggests that the mitochondrial P450 forms tested are not membrane-integral proteins. The mitochondrial-associated P450 forms are capable of metabolizing resorufin derivatives, erythromycin, and p-nitrophenol in an adrenodoxin- and adrenodoxin reductase-supported system. Treatment of rats with phenobarbital (PB) resulted in the induction of mitochondrial pentoxyresorufin O-deethylase (PROD), benzoxyresorufin O-deethylase (BROD), and erythromycin N-demethylase (ERND) activities by 17-, 23-, and 2-fold, respectively. These activities were inhibited by 33 to 64% by antibodies to P4502B1/2 and P4503A1/2. The induction of the above monooxygenase activities correlated with the levels of mitochondrial proteins cross-reacting with antibodies to P4502B1/2 and P4503A1/2 in PB-treated livers. Similarly, administration of beta-naphthoflavone (BNF) resulted in a marked elevation of O-deethylation of ethoxy-, benzoxy-, and methoxyresorufins and a 2-fold increase in ERND activity. Immunoblot and immunoinhibition experiments using P4501A1/2, P4502B1/2, P4503A1/2, and P4502E1 antibodies revealed the presence of P450 forms closely related to the microsomal inducible forms. Results of immunoinhibition studies, using antibodies to adrenodoxin and reconstitution of enzyme activity with purified P450 forms, suggested a role for the mitochondrial P450 in the metabolism of xenobiotic substrates. The purified mitochondrial P450s also exhibited overlapping substrate specificities for resorufin derivatives and erythromycin.

Xenobiotic metabolism in brain.

Recent hypothesis suggesting a role for environmental toxins in the pathogenesis of neurodegenerative disorders has stimulated interest in research on xenobiotic metabolizing capability of the brain. In addition to possible irreversible loss of neurons through bioactivation in situ in the nervous tissue, the metabolism of psychoactive drugs in the target tissue can lead to local pharmacological modulation at the site of action. The major drug metabolizing enzymes, cytochromes P-450 (P450) and flavin-containing monooxygenase (FMO) have been detected in rodent brain and human brain tissue obtained at autopsy. The brain microsomal and mitochondrial P450 systems are capable of metabolizing a variety of xenobiotics, while the brain FMO efficiently metabolizes a variety of psychoactive drugs to their respective N-oxides. Immunocytochemical studies have revealed the regional heterogeneity in the distribution of multiple forms of P450 in the brain and the co-localization of P450 and FMO predominantly in the neuronal cells. Although the brain P450 and FMO share many common features with similar enzymes present in other tissues such as liver and lung, there are some distinctive differences. It is evident from the studies carried out so far that the brain can metabolize a variety of lipophilic xenobiotics that enter by way of the blood stream.

Differential effect of chronic nicotine administration on brain cytochrome P4501A1/2 and P4502E1.

The effects of chronic nicotine administration on the enzymatic activities and immunoreactivities of P4501A1/2 and P4502E1 were determined in microsomal fractions from brain regions and liver. Chronic nicotine increased 7-ethoxyresorufin O-deethylase activity and P4501A1/2 immunoreactivity in hippocampus and brain stem, decreased values in cerebral cortex, thalamus, and striatum, and did not affect parameters in other brain regions. In contrast, N-nitrosodimethylamine N-demethylase activity from nicotine-injected rats increased in all brain regions examined, and this increase was associated with increased P4502E1 immunoreactivity. Chronic nicotine did not alter either enzyme activities or immunoreactivities of these isoforms in hepatic microsomes.

The chronic administration of nicotine induces cytochrome P450 in rat brain.

The objective of these studies was to determine whether chronic administration of nicotine altered the cytochrome P450 (P450) monooxygenase system in rat brain. Male Sprague-Dawley rats received injections of nicotine bitartrate (1.76 mg/kg, s.c., twice daily for 10 days), and total cytochrome P450 content, the activity of NADPH-cytochrome c reductase, and the activities and relative abundance of P4502B1 and P4502B2 (P4502B1/2) were determined in microsomal fractions from rat brain. The content of P450 increased significantly (p < 0.02) in all brain regions examined from nicotine-injected rats; the largest increase (208% of control) was in frontal cortex and the smallest increase (122% of control) in cerebellum. The activity of NADPH-cytochrome c reductase was unaltered by nicotine administration. Benzyloxyresorufin O-dealkylase (BROD) and pentoxyresorufin O-dealkylase (PROD) activities, mediated by P4502B1/2, increased significantly (p < 0.02) following nicotine administration; the largest increase (213-227% of control) was in frontal cortex. Western blots of microsomal proteins indicated that the increase in enzymatic activity was associated with an increase in content of P4502B1/2 immunoreactive proteins. In contrast to brain, total P450 content, activities of NADPH-cytochrome c reductase, BROD, and PROD, and levels of P4502B1/2 immunoreactive proteins in liver were unaffected by chronic nicotine administration. Results indicate that chronic nicotine administration regulates the expression of P4502B1/2 in brain and that at the dose schedule used this effect occurs without a demonstrable effect on the hepatic P450 monooxygenase system.

Induction of brain cytochrome P-450IIE1 by chronic ethanol treatment.

Cytochrome P-450 mediated metabolism is potentially involved in the expression of the pharmacological and/or toxicological effects of a wide variety of drugs and environmental chemicals upon tissues which contain this metabolic system. In the present investigation, the presence of cytochrome P-450IIE1 and associated mono-oxygenase activities in brain and the effect of chronic ethanol treatment on brain cytochrome P-450 (P-450) were studied. Aniline hydroxylase, N-nitroso-dimethylamine N-demethylase and p-nitrophenol hydroxylase activities (known to be mediated by P-450IIE1) were detectable in brain microsomes from untreated rats and were about 5%, 125% and 8.3%, respectively, of the corresponding hepatic levels. Chronic ethanol treatment resulted in induction of the above enzyme activities in brain microsomes by 243%, 496% and 155%, respectively. Intake of ethanol for a prolonged period also resulted in the induction of total P-450 in the brain (150% of the control). Addition of the antisera raised against rat liver cytochrome P-450IIE1 markedly inhibited brain microsomal p-nitrophenol hydroxylase activity. Immunoblot analysis of rat brain microsomes using the above antisera also revealed the induction of brain cytochrome P-450IIE1 following chronic ethanol administration. Immunocytochemical localization of cytochrome P-450IIE1 using the above antisera, revealed the preferential localization of the enzyme in the neuronal cell bodies in the cortex, hippocampus, basal ganglia, hypothalamic nuclei and reticular nuclei in the brainstem of rats treated chronically with ethanol. Based upon these studies, it is conceivable that chronic alcohol ingestion could enhance the sensitivity of certain regions of the brain to environmental chemicals that are metabolized to more toxic derivatives by the P-450 system.

Characterization of a phenobarbital-inducible cytochrome P-450, NADPH-cytochrome P-450 reductase and reconstituted cytochrome P-450 mono-oxygenase system from rat brain. Evidence for constitutive presence in rat and human brain.

Cytochrome P-450 was purified to apparent homogeneity from the brain microsomes of phenobarbital-treated rats. The specific content of the purified P-450 was 12.7 nmol/mg of protein. NADPH-cytochrome P-450 reductase (reductase) was also purified to apparent homogeneity from brain microsomes. The specific content was 34.7 mumol of cytochrome c reduced/min per mg of protein. The reduced carbon monoxide spectrum of purified P-450 exhibited a peak at 450 nm. Both the P-450 and the reductase moved as single bands on SDS/PAGE. The molecular masses of the purified P-450 and the reductase were determined to be 53.3 and 72.0 kDa respectively. The purified brain P-450 cross-reacted with antibodies to rat liver P-450IIB1/IIB2 when examined by Western immunoblotting, but no immunological similarity was observed with rat liver P-450IA1/IA2 or P-450IIE1. Purified rat brain reductase cross-reacted with antibodies to rat liver reductase. Further, immunoblot experiments with untreated rat and human brain microsomes using antisera to the purified rat brain P-450 and reductase indicated that these forms of P-450 and NADPH-cytochrome P-450 reductase exist constitutively in rat and human brain. Purified rat brain P-450 was reconstituted with purified NADPH-cytochrome P-450 reductase, deoxycholate and dilauroyl glyceryl 3-phosphocholine. NADPH-dependent N-demethylation of aminopyrine and morphine was observed in the reconstituted system. The catalytic-centre activities were 80.25 and 38.2 nmol of formaldehyde formed/min per nmol of P-450 respectively. The reconstituted system had a comparatively lower catalytic-centre activity for 7-ethoxycoumarin O-de-ethylase (10.5 nmol of product formed/min per nmol of P-450).