Toshio Yasumori

Human MDR1 polymorphism : G2677T/A and C3435T have no effect on MDR1 transport activities

The two most frequently observed single nucleotide polymorphisms (SNPs) of the human multidrug resistance 1 (MDR1) gene are 2677G/T/A (893Ala/Ser/Thr) and 3435C/T (no amino acid substitution). In this study, six forms of MDR1 cDNAs with the SNPs were expressed in LLC-PK1 cells and their transport activities were determined. Nearly identical amounts of the recombinant MDR1 proteins were expressed in the established cell lines using the Flp recombinase, which integrates a gene of interest at a specific genomic location. Four structurally diverse compounds: verapamil, digoxin, vinblastine and cyclosporin A, were examined for transcellular transport activities and intracellular accumulation. No significant differences were observed between cells expressing five polymorphic types of the MDR1 cDNAs (2677G/3435T, 2677A/3435C, 2677A/3435T, 2677T/3435C, 2677T/3435T) and cells expressing the wild-type (2677G/3435C). These results suggested that the two frequently observed MDR1 SNPs had no effect on the transport activities of MDR1 proteins expressed in LLC-PK1 cells in vitro, and other genetic or environmental factors might control the expression of MDR1 and the in vivo activity of MDR1.

Polymorphism in hydroxylation of mephenytoin and hexobarbital stereoisomers in relation to hepatic P-450 human-2

Stereoselective 4′‐hydroxylations of R‐(−)‐mephenytoin and S‐(+)‐mephenytoin and 3′‐hydroxylation of R‐(‐)‐hexobarbital and S‐(+)‐hexobarbital were determined in liver microsomes of 14 Japanese subjects who were extensive metabolizers of mephenytoin and in five Japanese subjects who were poor metabolizers of mephenytoin. Content of P‐450 human‐2 assessed by Western blots was correlated to microsomal S‐(+)‐mephenytoin 4′‐hydroxylation, R‐(−)‐hexobarbital 3′ α‐hydroxylation, and S‐(+)‐hexobarbital 3′ ß‐hydroxylation, and was less correlated to R‐(−)‐mephenytoin 4′‐hydroxylation, R‐(−)‐hexobarbital 3′ ß‐hydroxylation, and S‐(+)‐hexobarbital 3′ α‐hydroxylation. Antibodies raised against P‐450 human‐2 inhibited microsomal S‐(+)‐mephenytoin 4′‐hydroxylation efficiently but was less efficient on R‐(−) ‐mephenytoin 4′‐hydroxylation in extensive metabolizers and on 4′‐hydroxylation of mephenytoin enantiomers in poor metabolizers. The antibodies also inhibited R‐(−)‐hexobarbital 3′ α‐hydroxylation and S‐(+)‐hexobarbital 3′ ß‐hydroxylation but did not effectively inhibit the hydroxylation of the two other optical isomers of hexobarbital in extensive metabolizers and of four stereoisomers in poor metabolizers. These findings indicate the close relationship between polymorphic mephenytoin 4′‐hydroxylation and two stereospecific hexobarbital hydroxylations, and they suggest that P‐450 human‐2 is a typical S‐(+)‐mephenytoin 4′‐hydroxylase and a major hexobarbital 3′‐hydroxylase in the livers of extensive metabolizers. The findings were further supported by the experiments that used P‐450 human‐2 complementary dexoyribonucleic acid‐derived protein in yeast microscomes.

Endothelin-1 inhibits induction of nitric oxide synthase and GTP cyclohydrolase I in rat mesangial cells.

To investigate the interaction between endothelin (ET) and the nitric oxide system, we examined the effects of ET-1 and ET-3 on the induction of inducible nitric oxide synthase (iNOS) and guanosine triphosphate cyclohydrolase I (GTP:CHI), the rate-limiting enzyme of de novo synthesis of the cofactor tetrahydrobiopterin (BH4), in rat mesangial cells. ET-1 inhibited the nitrite accumulation induced by a combination of interleukin-1 beta, tumor necrosis factor-alpha, and lipopolysaccharide in a concentration-dependent manner. The inhibitory effect of ET-3 was less potent than that of ET-1. A selective ETA antagonist, BQ-485, and an ETA and ETB antagonist, TAK-044, abolished the inhibitory effects of ET-1, whereas the selective ETB antagonist BQ-788 had no effect on the inhibition produced by ET-1. These observations indicate that ET-1 inhibits cytokine-stimulated nitrite accumulation through the ETA receptor. Western blot analysis showed that the suppression of nitrite accumulation was accompanied by a decrease in iNOS protein. Northern blot analysis showed that ET-1 inhibited the expression of both iNOS and GTP:CHI mRNA. In conclusion, ET-1 inhibits cytokine-stimulated nitric oxide production through the ETA receptor by suppressing the expression of iNOS and GTP:CHI mRNA in rat mesangial cells.

Characterization of human P450IIC isozymes by using yeast expression system.

Publisher Summary This chapter discusses the characterization of human P450IIC isozymes by using yeast expression system. As in experimental animal species, multiple forms of P450IIC isozymes are expressed in human livers, and these isozymes catalyze polymorphic hydroxylations of the clinically important drugs mephenytoin and tolbutamide. The isozymes in humans can be divided into two subgroups, IIC8 and IIC9/10, based on differences in amino-terminal protein sequences and apparent molecular weights on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Purified P450IIC9/10 preparations reported from several laboratories showed differences in catalytic activities but identical amino-terminal protein sequences, and these were reported to be indistinguishable by extensive chemical analysis.