Aspirin and salicylic acid do not inhibit methyl jasmonate-inducible expression of a gene for ornithine decarboxylase in tobacco BY-2 cells.

Similar to the prostanoid-mediated inflammatory response in mammals, jasmonate-mediated wound response in plant leaves is inhibited by salicylic acid (SA) or acetylsalicylate (aspirin). In tobacco BY-2 cells, expression of the gene for ornithine decarboxylase (ODC) involved in putrescine synthesis is rapidly inducible by methyl jasmonate (MeJA). A nuclear gene for ODC isolated from tobacco, gNtODC-1, was an intron-less gene and MeJA induced the expression of a GUS fusion gene with the gNtODC-1 promoter in transformed tobacco cells. Although SA alone did not induce the expression, 0.2 to 20 microM SA increased the MeJA-induced expression of the fusion gene to about two-fold. A similar increase was observed with aspirin but not with 3- or 4-hydroxybenzoic acids. SA at concentrations up to 200 microM did not inhibit the MeJA-induction of mRNAs for the GUS fusion gene and the endogenous gene for ODC.

Comparisons between in-vitro and in-vivo metabolism of (S)-warfarin: catalytic activities of cDNA-expressed CYP2C9, its Leu359 variant and their mixture versus unbound clearance in patients with the corresponding CYP2C9 genotypes.

To study whether an in-vitro model for three different genotypes of human CYP2C9*3 polymorphism would be useful for predicting the in-vivo kinetics of (S)-warfarin in patients with the corresponding genotypes, the intrinsic clearance (Cl(int) or Vmax/Km) for (S)-warfarin 7-hydroxylation obtained from recombinant human CYP2C9*1 [wild-type (wt)] and CYP2C9*3 (Leu359/Leu) expressed in yeast and the mixture of equal amounts of these were compared with the in-vivo unbound oral CI (CI(po,u)) of (S)-warfarin obtained from 47 Japanese cardiac patients with the corresponding CYP2C9 genotypes. The in-vitro study revealed that the recombinant CYP2C9*1 (wt/wt), 2C9*3 (Leu359/Leu) and their mixture (Ile359/Leu) possessed a mean Km of 2.6, 10.4 and 6.6 microM and Vmax of 280, 67 and 246 pmol/min/nmol P450, respectively. Thus, the mean in-vitro Cl(int) obtained from recombinant CYP2C9*3 (Leu359/Leu) and the mixture (Ile359/Leu) of 2C9*3 and 2C9*1 were 94% and 65% lower than that obtained from CYP2C9*1 (wt/wt) (6.7 versus 38 versus 108 ml/min/micromol P450, respectively). The in-vivo study showed that the median Cl(po,u) for (S)-warfarin obtained from patients with homozygous (Leu359/Leu, n = 1) and heterozygous (Ile359/Leu, n = 4) CYP2C9*3 mutations were reduced by 90% (62 ml/min) and 66% (212 ml/min, P < 0.05) compared with that obtained from those with homozygous 2C9*1 (625 ml/min, n = 42). Consequently, there was a significant correlation (r = 0.99, P < 0.05) between the in-vitro Cl(int) for (S)-warfarin 7-hydroxylation and the in-vivo Cl(po,u) for (S)-warfarin in relation to the CYP2C9*3 polymorphism. In conclusion, the in-vitro model for human CYP2C9*3 polymorphism using recombinant cytochrome P450 proteins would serve as a useful means for predicting changes in in-vivo kinetics for (S)-warfarin and possibly other CYP2C9 substrates in relation to CYP2C9*3 polymorphism.

Metabolism of warfarin enantiomers in Japanese patients with heart disease having different CYP2C9 and CYP2C19 genotypes.

OBJECTIVE: To determine whether genetic polymorphism of cytochrome P450 (CYP) 2C9 affects the in vivo metabolism of warfarin enantiomers. METHODS: Eighty-six Japanese patients heart disease who were given warfarin participated in the study. Plasma unbound concentrations of warfarin enantiomers and urinary (S)-7-hydroxywarfarin concentrations were measured by means of a chiral HPLC and ultrafiltration technique to calculate the unbound oral clearance (CLpo,u) for the enantiomers and the formation clearance (CLm) for (S)-warfarin 7-hydroxylation. Genotyping for CYP2C9 (the wild type [wt], Arg144/Cys, and I1e359/Leu) and for CYP2C19 (wt, ml, and m2) was performed with a polymerase chain reaction method. RESULTS: Three patients were heterozygous for the CYP2C9 Leu359 mutation but none were homozygous for the mutation (the allele frequency of 0.017). None had a CYP2C9 Cys144 allele. The medians for (S)-warfarin CLpo,u and its 7-hydroxylation CLm obtained from heterozygotes of CYP2C9 Leu359 were significantly less than those obtained from homozygotes of the wt allele, as follows: 234 ml/min (range, 156 to 269 ml/min) versus 632 ml/min (range, 180 to 2070 ml/min) (p < 0.001) and 0.20 ml/min (range, 0.05 to 0.77 ml/min) versus 0.80 ml/min (range, 0.05 to 14.9 ml/min) (p < 0.05), respectively. In contrast, no difference was observed in (R)-warfarin CLpo,u between the groups. The allele frequencies for CYP2C19 m1 and CYP2C19 m2 were 0.26 and 0.14, respectively, indicating 15% of patients were genotypically poor metabolizers of CYP2C19. No difference in CLpo,u for warfarin enantiomers was observed between the assumed CYP2C19 phenotypes. CONCLUSION: Heterozygotes for CYP2C9 I1e359/Leu allele have reduced in vivo metabolism of (S)-warfarin but not (R)-warfarin. Because (S)-warfarin has a greater anticoagulant potency than its (R)-congener, the genetic polymorphism of CYP2C9 may partly account for the large interpatient variability in therapeutic dosages of warfarin.

Determination of unbound warfarin enantiomers in human plasma and 7-hydroxywarfarin in human urine by chiral stationary-phase liquid chromatography with ultraviolet or fluorescence and on-line circular dichroism detection.

Enantiomers of warfarin and 7-hydroxywarfarin in human plasma and urine, respectively, were determined by high-performance liquid chromatography using a cellulose-derivative column with UV or fluorescent detection, and their absolute configuration was determined simultaneously by a circular dichroism spectropolarimeter connected in series. Enantiomers of warfarin and its major metabolites [i.e., (R)-6-hydroxywarfarin, (S)-7-hydroxywarfarin and (RS)-warfarin alcohol] were well resolved. The method was precise and sensitive: within- and between-day coefficients of variation were <9.6% for warfarin enantiomers in plasma and <7.1% for 7-hydroxywarfarin enantiomers in urine, respectively, and the lower detection limits were 20 ng/ml for (R)-warfarin, 40 ng/ml for (S)-warfarin, 2.5 ng/ml for (R)-7-hydroxywarfarin and 4.5 ng/ml for (S)-7-hydroxywarfarin in 0.5 ml of both plasma and urine. The ultrafiltration technique was used for determining unbound concentrations of warfarin enantiomers in plasma using [14C]warfarin enantiomers resolved by the present HPLC system. Clinical applicability of the method was evaluated by determining unbound concentrations of warfarin enantiomers in five consecutive plasma samples obtained from a patient exhibiting an unstable anticoagulant response to warfarin (4 mg/day, p.o.). Results indicated that the present method would be useful in clarifying factors responsible for a large intra- and inter-patient variability in warfarin effects with regard to unbound plasma enantiomer pharmacokinetics.

Cloning, functional expression and tissue distribution of human alpha 1c-adrenoceptor splice variants.

We report the cloning and characterization of two isoforms of human alpha 1c-adrenoceptor cDNA (alpha 1c-2, alpha 1c-3). These isoforms are generated by alternative splicing and differ from the clone we previously isolated (alpha 1c-1) in their length and sequences of the C-terminal domain. Tissue distribution of mRNAs showed that these variants co-express with alpha 1c-1 in the human heart, liver, cerebellum and cerebrum. Despite the structural differences, functional experiments in transfected CHO cells showed that the three isoforms have similar ligand binding properties, and all couple with phospholipase C/Ca2+ signaling pathway.