Joan E. Clark

20-Hydroxyeicosatetraenoic acid is an endogenous vasoconstrictor of canine renal arcuate arteries

Recent studies have indicated that renal arteries can produce 20-hydroxyeicosatetraenoic acid (20-HETE) and suggest the potential involvement of a P450 metabolite of arachidonic acid in the myogenic activation of canine renal arteries. In the present study, the effects of 20-HETE on isolated canine renal arcuate arteries were studied. Administration of 20-HETE to the bath or the lumen at concentrations of 0.01-1 microM produced a graded reduction in the diameter of these vessels. In contrast, 19(R)-HETE was a vasodilator, whereas 19(S)-HETE was relatively inactive. The vasoconstrictor response to 20-HETE was not altered by the cyclooxygenase inhibitor indomethacin, endoperoxide/thromboxane receptor antagonist SQ29548, or combined blockade of the cyclooxygenase, lipoxygenase, and P450 pathways using indomethacin, baicalein, and 7-ethoxyresorufin. The response to 20-HETE was associated with depolarization and a sustained increase in the intracellular calcium concentration in renal vascular smooth muscle cells. Patch-clamp studies indicated that 20-HETE significantly reduced mean open time, the open-state probability, and the frequency of opening of a 117-pS K+ channel recorded from renal vascular smooth muscle cells in the cell-attached mode. Microsomes prepared from the renal cortex of dogs produced 20-HETE and 20-carboxyarachidonic acid when incubated with [14C]arachidonic acid. These results indicate that 20-HETE is an endogenous constrictor of canine renal arcuate arteries. The vasoconstrictor response to 20-HETE resembles the myogenic activation of these vessels after elevations in transmural pressure and suggests a potential role for this substance in the regulation of renal vascular tone.

Inhibitors of cytochrome P-450 attenuate the myogenic response of dog renal arcuate arteries.

The role of cytochrome P-450 in the myogenic response of isolated, perfused renal arcuate arteries of dogs to elevations in transmural pressure was examined. The phospholipase A2 inhibitor oleyloxyethylphosphorylcholine (1 and 10 microM) inhibited the greater than threefold increase in active wall tension in these arteries after an elevation in perfusion pressure from 80 to 160 mm Hg. Inhibition of cyclooxygenase activity with indomethacin (1 or 10 microM) had no effect on this response. The cytochrome P-450 inhibitors ketoconazole (10 and 100 microM) and beta-diethyl-aminoethyldiphenylpropylacetate (SKF 525A, 10 and 100 microM) also inhibited the myogenic response. At a pressure of 160 mm Hg, SKF 525A (10 microM) and ketoconazole (100 microM) reduced active wall tension in renal arteries by approximately 70%. Partial inhibition of the myogenic response was obtained after perfusion of the vessels with mechanism-based inhibitors of P-450, 1-aminobenzotriazole (75 microM) and 12-hydroxy-16-heptadecynoic acid (20 microM). The thromboxane receptor antagonist SQ 29,548 (1 or 10 microM) had no effect on the pressure-induced increase in active wall tension in renal arteries. Arachidonic acid (50 microM) constricted isolated perfused renal arteries and potentiated the myogenic response in the presence of indomethacin. This response was completely reversed by ketoconazole (100 microM) or SKF 525A (100 microM). Microsomes (1 mg/ml) prepared from small renal arteries (200-500 microns) and incubated with [1-14C]arachidonic acid (0.5 mu Ci, 50 microM) produced a metabolite that coeluted with 20-hydroxyeicosatetraenoic acid (20-HETE) during reversed-phase high-performance liquid chromatography. The formation of this product was inhibited by both ketoconazole and SKF 525A at concentrations of 10 and 100 microM. These results are consistent with the involvement of the vasoconstrictor 20-HETE and other cytochrome P-450 metabolites of endogenous fatty acids in the myogenic response.

Characterization of a cDNA encoding a human kidney, cytochrome P-450 4A fatty acid ω-hydroxylase and the cognate enzyme expressed in Escherichia coli

A cDNA encoding a cytochrome P-450 4A (CYP4AII) was cloned from a human kidney cDNA library. Northern blot analysis and RNase protection assays indicate that related mRNAs occur in kidney and liver with the highest abundance found in kidney. The enzyme was expressed from its cDNA in Escherichia coli. A solubilized preparation of the enzyme reconstituted with cytochrome P-450 reductase catalyzed the omega-hydroxylation of lauric acid, palmitic acid, and arachidonic acid with turnover numbers of 9.8, 2.2 and 0.55 min-1, respectively. Little or no activity was detected toward prostaglandins A1 and E1.

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

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.