Structure-Function Studies and Physiological Roles of Eicosanoids Metabolized by Cytochrome P450 ω-Hydroxylases

Abstract

The cytochrome P450-mediated oxygenation of many fatty acids and eicosanoids occurs in a variety of tissues. The cytochromes P450 responsible for these activities belong to a closely related gene family, the first member of which was purified and characterized from clofibrate-treated rats by Tamburini, et al. (1) and cloned and sequenced by Hardwick, et al (2). In the latest listing of members of the cytochrome P450 gene superfamily by Nebert, et al. (3), there are now 9 members of the CYP4A gene subfamily which includes those cytochromes P450 catalyzing the ω-hydroxylation of fatty acids and eicosanoids from different organs and species. Since the mid-1970’s, the interest of Masters’ laboratory has centered on fatty acid and prostaglandin hydroxylations catalyzed by pig and rabbit kidney and lung microsomes. Following the observation by Powell and Solomon (4) and Powell (5) that the ω-hydroxylation of prostaglandins and thromboxanes was induced in maternal rabbit lung over 100-fold during pregnancy, Williams, et al. (6) purified a cytochrome P450 prostaglandin w-hydroxylase from this source. Simultaneously, Yamamoto, et al. (7) isolated a similar or identical protein from the lungs of progesterone-treated rabbits and, later, Matsubara, et al (8) cloned and sequenced the cDNA for their protein. There is strong evidence that the high level of induction of this enzyme is regulated at the transcriptional level, since the gestational age dependence of enzyme activity parallels the levels of protein (Western blots) and in vitro-translatable mRNA (9). Northern blotting techniques have also revealed an increase in mRNA levels as a result of pregnancy in female rabbits or progesterone treatment in male rabbits (8,10). Recently, attention has been directed toward the possible physiological significance of these enzymes (11–15) especially in the regulation of hemodynamics. The experiments to be described have been designed to ascertain the structure-function relationships of the various members of the CYP4A gene subfamily by correlating the various substrate specificities with manipulation of their amino acid sequences and expression in a transient mammalian cell expression system, i.e., African green monkey kidney cells (COS-1). The regulatory aspects of these many and varied, but closely related, enzymes are being studied in whole animals by pretreatment with steroid hormones, some of which have been shown previously to be inducers of various members of this subfamily. The physiological roles of these various eicosanoid-metabolizing cytochromes P450 are being probed utilizing a combination of biochemical approaches, including the use of broad spectrum and specific inhibitors of cytochrome P450 systems in microdissected renal arcuate arteries.