Incubation of cocultures of human aortic endothelial (HAEC) and smooth muscle cells (HASMC)

A cytochrome P-450 (P-450) multigene family codes for several related human liver enzymes, including the P-450 responsible for (S)-mephenytoin 4'-hydroxylation. This enzyme activity has previously been shown to be associated with a genetic polymorphism. Genomic (Southern) blot analysis using non-overlapping 5' and 3' portions of a cDNA clone suggests that approximately seven related sequences are present in this gene family. In this study four cDNA clones, all nearly full-length, were isolated from a bacteriophage lambda gt11 library prepared from a single human liver. These clones can be grouped into two categories that are approximately 85% identical at the level of DNA sequence. The cDNA clones in one category (MP-4, MP-8) both match the N-terminal sequences of the P-450MP-1 and P-450MP-2 proteins, which had previously been shown to be catalytically active in (S)-mephenytoin 4'-hydroxylation. These two cDNAs, MP-4 and MP-8, differ in only two bases in the coding region but are quite distinct in their 3' noncoding regions. Another protein (P-450MP-3) was isolated on the basis of its immunochemical similarity to P-450MP-1 but was found to be catalytically inactive; amino acid sequencing of tryptic peptides of P-450MP-3 showed a correspondence to the second category of cDNA clones (MP-12, MP-20), which differ from each other in only four (nonsilent) base changes. Oligonucleotides specific for the two groups of cDNA clones were used as probes of human liver mRNAs--individual liver samples examined expressed both types of mRNAs but no correlation was observed between the abundance levels of any mRNA and catalytic activity. Further, oligonucleotide probes indicated that mRNAs corresponding to both the MP-4 and MP-8 clones were apparently present in individual liver samples. A monoclonal antibody was isolated that recognized P-450MP-1 but not P-450MP-2 or P-450MP-3; the amount of protein detected by the antibody in different liver samples was not correlated with the mephenytoin 4'-hydroxylase activity. These results indicate that several closely related P-450 genes are all expressed in individual human livers. The MP-4/MP-8 gene products are proposed to be the ones most likely involved in mephenytoin 4'-hydroxylation, and much of the variation in catalytic activity among individuals is not a result of differences in levels of P-450MP-1 or mRNA but may be due to base differences in the structural gene(s).

Human liver cytochrome P-450NF is the form of cytochrome P-450 responsible for the oxidation of the calcium-channel blocker nifedipine, which has been reported to show polymorphism in clinical studies. By screening a bacteriophage Xgtll expression cDNA library, we isolated two clones: NF95 with an insert length of 0.8 kilobases which gave a stable fusion protein and NF25 with an insert length of 2.2 kilobases. The two clones were both sequenced and shown to be identical in their overlapping section. The sequence of NF25 is 77% similar to that reported for a rat cytochrome '"P-45OPCN" cDNA (PCN = pregnenolone-16a-carbonitrile). The similarity decreases to 45-53% when the sequence is compared to human cytochromes P-450 belonging to other families [i.e., "pH P-450(1)," "P1-450," "P3-450," and "P-45OMP." The deduced amino acid sequence is 73% similar to that of rat cytochrome P-45OPCN, and the first 21 amino acids are identical to those reported for human liver cytochrome "P-450p." Sections of these clones were nick-translated and used as probes for analyses of human mRNA and genomic DNA. The number and size of bands indicate that P-45ONF belongs to a multigene family, the so-called pregnenolone-16a-carbonitrile-inducible family.Cytochrome P-450 (P-450) plays an important role in the oxidation of drugs and carcinogens as well as endogenous substrates. Interindividual variation in oxidative metabolism can be attributed, at least in part, to the composition of individual P-450 forms, and these differences are partly due to genetic factors. Since 1977, several genetic polymorphisms of drug oxidation have been demonstrated (1-3), and in some cases the involved form of P-450 has been identified in humans (4, 5). Recently Kleinbloesem et al. (6) reported that oxidation of nifedipine, a vasodilator and calcium-channel blocker, was distributed in a polymorphic manner-17% of the Dutch population studied were phenotypically poor metabolizers. Recently we identified and purified the human liver P-450 form that is responsible for this oxidation of nifedipine (7). This protein, P-45ONF, was shown to be related or identical to P-450s previously isolated from human liver in this laboratory (8). To better understand the mechanism underlying this polymorphism, we used polyclonal and monoclonal antibodies to screen a human liver bacteriophage Xgtll cDNA expression library. The selected clones were analyzed, sequenced, and used to prepare nick-translated probes for the analysis of mRNA and genomic DNA. MATERIALS AND METHODSEnzymes and Antibodies. Human livers were obtained through the Nashville Regional Organ Procurement Agency, and protocols were approved by the Vanderbilt Committee for the Protection of Human Subjects. Livers were perfused immediately after circulatory arrest, chilled on ice, and brought to the laboratory. The livers were cut in small pieces, frozen in liquid nitrogen, and stored at -70°C (8).P-45ONF was purified as described, and polyclonal and monoclonal antibodies were produced (7, 9). These antibodies r...

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