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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.

ABSTRACT: 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).

SUBMITTER: Anandatheerthavarada HK

PROVIDER: S-EPMC1132036 | biostudies-other | 1992 Dec

REPOSITORIES: biostudies-other

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Project description:Cyclo-oxygenases (COXs) catalyse the synthesis of PGH2 (prostaglandin H2), which serves as the common substrate for the production of PGE2, PGD2, PGF(2alpha), prostacyclin (or PGI2) and TXs (thromboxanes). While COX-1 is the major isoform responsible for prostanoid synthesis in healthy tissues, little information is available on the contribution of constitutive COX-2 to the various prostanoid synthetic pathways under non-inflammatory conditions. To evaluate further the role of COX-2 in prostanoid biosynthesis, rats were acutely treated with the selective COX-1 inhibitor SC-560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole] or the selective COX-2 inhibitors MF tricyclic [3-(3,4-difluorophenyl)-4-(4-(methylsulphonyl)phenyl)-2-(5H)-furanone] and DFU [5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2-(5H)-furanone]. Selected tissues were then processed for a complete analysis of their prostanoid content by liquid chromatography MS. Whereas the treatment with SC-560 caused a 60-70% inhibition in the total prostanoid content of most tissues examined, a significant decrease (35-50%) in total prostanoid content following selective COX-2 inhibition was solely detected for kidney and brain tissues. Analysis of the individual prostanoids reveals significant inhibition of 6-oxo-PGF(1alpha), PGE2, PGD2, PGF(2alpha) and TXB2 in the kidney and inhibition of all these prostanoids with the exception of PGD2 in the forebrain. These results demonstrate that constitutively expressed COX-2 contributes to the production of prostanoids in kidney and brain for each of the PGE2, PGI2 and TXB2 pathways under non-inflammatory conditions. Approaches to modulate inflammation through specific inhibition of terminal synthases, such as mPGES-1 (microsomal PGE2 synthase-1), thus have the potential to differ from COX-2 inhibitors and non-selective non-steroidal anti-inflammatory drugs with regard to effects on constitutive prostanoid synthesis and on renal function.

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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.

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