Characterization of an endogenous inhibitor of lung 15-hydroxyprostaglandin dehydrogenase.

An endogenous inhibitor of the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase was isolated from the 105,000 X g supernatant fraction of lungs of pregnant rabbits following DEAE chromatography. The material was heat stable and was resistant to pronase treatment. The inhibitor contained a mixture of saturated and mono-unsaturated fatty acids and cholesterol with palmitate and oleate representing the major fatty acids in the inhibitory factor. The factor inhibited prostaglandin dehydrogenase activity but had only minor effects on the activity of NAD+-dependent alcohol and lactate dehydrogenases or the NADP+-dependent isocitrate dehydrogenase. In an attempt to develop a greater understanding of the inhibitory action of fatty acids on prostaglandin dehydrogenase activity, a variety of standard fatty acids were examined for their ability to decrease enzymic activity. Oleate and palmitate inhibited enzymic activity by 70% at 10 microM, whereas arachidonate and myristate were only 30% inhibitory at this concentration. A comparison among the 18-carbon-containing fatty acids demonstrated that oleate was more potent than linoleate and linolenate in inhibiting prostaglandin dehydrogenase activity. The coenzyme A derivatives of oleate, linoleate and linolenate were less inhibitory than the free fatty acids.

Cytochrome P450 4A fatty acid omega hydroxylases.

The Cytochrome P450 4A subfamily is one of eighteen subfamilies in the CYP4 family and presently consists of twenty individual forms in nine different mammalian species. The major substrates for CYP4A forms are fatty acids, but recent studies have shown other non-fatty acid substrates may be metabolized by specific CYP4A forms. The physiological and metabolic functions of the CYP4A subfamily have not been elucidated, but the ability of CYP4A forms to metabolize medium and long chain length fatty acids at their omega (omega)-carbon atom has generated significant interest because of the possible role that omega-hydroxylated fatty acids may have in cell signalling processes and as an alternative pathway for fatty acid metabolism. A number of different compounds or physiological conditions have been shown to regulate the expression of CYP4A forms in liver and/or kidney. Several CYP4A forms may serve as a marker for the exposure to compounds that are classified as peroxisome proliferators. There is also considerable interest why multiple CYP4A forms exist in different tissues. Recent studies in the rat and human indicate that other CYP4 forms besides CYP4A forms may be responsible for the metabolism of arachidonic acid to its omega-hydroxy product. The focus of this review will be to summarize recent studies that have characterized the substrate specificity of rat, rabbit and human CYP4A forms and discuss the significance of CYP4A-mediated hydroxylation of fatty acids. In addition, dietary effects or novel compounds that have been reported to regulate CYP4A expression in the rat and mouse will be discussed.

Changes in ovarian NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase activity in pregnant and pseudopregnant rabbits.

The specific activity of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to increase in the ovaries of pregnant and pseudopregnant rabbits. The mean specific activity of cytosolic ovarian PGDH in 14- to 28-day pregnant rabbits was 24.3 +/- 8.1 nmol NADH formed/min/mg protein (n = 16) using PGE1 as substrate whereas in nonpregnant rabbits the specific activity was 1.5 +/- 0.8 nmol NADH formed/min/mg protein (n = 8). The reaction was dependent on NAD+; NADP+ did not support the reaction. In grouping the PGDH activities from pregnant rabbits into second (14-18 days) and third (2-28 days) trimester periods, no significant difference between values was found (26.1 +/- 8.9 vs 23.4 +/- 8.1 nmol NADH formed/min/mg protein, respectively). Western blot analysis of the ovarian cytosol using an antibody which was made to the purified lung PGDH of pregnant rabbits recognized an ovarian protein of identical molecular mass (30 kDa). Ovarian PGDH activities were also examined in rabbits treated with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to induce a state of superovulatory/pseudopregnancy and only on day 11 following hCG treatment was an increase in PGDH specific activity observed. On day 11, the specific activity was 14.8 +/- 4.3 nmol NADH formed/min/mg protein whereas values on days 10 and 12 were only 1.1 +/- 1.1 and 1.0 +/- 0.8, respectively. PGDH activities on days 3, 7 and 16 were also low.(ABSTRACT TRUNCATED AT 250 WORDS)

Perinatal changes in choroidal 15-hydroxyprostaglandin dehydrogenase: implications for prostaglandin removal from brain.

We have previously shown in the sheep fetus at 0.7 and 0.9 gestation that the choroid plexus, unlike brain parenchyma, catabolizes prostaglandins (PGs). Peculiarly, in the choroid plexus, PGE(2) catabolism persists throughout the neonatal period to abate in the adult, while PGF(2alpha) catabolism abates shortly after birth. To explain this differential behavior and elucidate the function of catabolic enzymes, we examined the cellular location and activity of the rate-limiting enzyme for PGE(2) and PGF(2alpha) catabolism, 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Immunofluorescence histochemistry and immunogold electronmicroscopy revealed abundant 15-PGDH expression in the epithelial cytosol close to the brush-border membrane at 0.7 and 0.9 gestation. In contrast, at 5 and 15 days postnatal, 15-PGDH was found throughout the cytosol of stromal fibroblasts. No staining was observed at either location in pregnant adults. PGF(2alpha) catabolism was minimal in the total homogenate and 100000xg supernatant of the fetal choroid plexus at 0.7 and 0.9 gestation, while PGE(2) catabolism was evident at 0.7 gestation only. In contrast, both PGs were catabolized in minced specimens at either age. In conclusion, our study shows immunoreactive 15-PGDH in the choroid plexus from fetal and neonatal, but not pregnant adult, sheep. Results suggest that PGE(2) catabolism is not as critically dependent as that of PGF(2alpha) on tissue integrity and 15-PGDH location. Given the key role being assigned to the choroid plexus in PG removal from brain, we speculate that persistence of PGE(2) catabolism into the early postnatal period protects against central respiratory depression caused by the compound during this susceptible stage of development.

Modification of lipoperoxidative effects of dichloroacetate and trichloroacetate is associated with peroxisome proliferation.

Pretreatment of male B6C3F1 mice with clofibric acid (CFA) or trichloroacetic acid (TCA) in the drinking water results in a marked decrease in the lipoperoxidative response as measured by the production of thiobarbituric acid reactive substances (TBARS) in mouse liver homogenates following acute dosing with TCA or dichloroacetic acid (DCA). Pretreatment with TCA or CFA also increased palmitoyl-CoA oxidase activity, microsomal 12-(omega) hydroxylation of lauric acid and expression of P450 4A isoforms. At the doses utilized, DCA-pretreatment did not increase the level of P450 4A protein, or markers of peroxisome proliferation. However, DCA-pretreatment did result in enhanced levels of TBARS, following acute dosing with DCA, compared to controls. Pretreatment with DCA, TCA, or CFA did not alter p-nitrophenol hydroxylation (an assay specific for P450 2E1), and no increases in immunodetectable P450 2E1, 4A, 1A1/2, 2B1/2 or 3A1 protein were observed. Assays from CFA- and TCA-pretreated mice suggest that the reduction in the TBARS response seen in TCA-pretreated animals results from activities associated with peroxisome proliferation. This might result from the induction of systems efficient in scavenging of peroxide intermediates or detoxification of aldehyde by-products of lipid peroxidation.

Characterization of a cytochrome P450 from di(2-ethylhexyl) phthalate-treated rats which hydroxylates fatty acids.

A cytochrome P450 was purified from liver microsomes of rats treated with di(2-ethylhexyl) phthalate (DEHP). DEHP is a member of a group of structurally diverse compounds which have been classified as peroxisome proliferators and are inducers of cytochromes P450 which hydroxylate lauric acid and other fatty acids. The P450 isolated from DEHP-treated rats (P450DEHP) was observed to have a Mr value of 51 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a maximum absorbance of 452 nm in its reduced carbon monoxide bound state. The amino terminal residue for P450DEHP was alanine and an 18-amino acid segment at the N-terminal region was identified. The N-terminal amino acid for the P450 4A1 from clofibrate-treated rats is methionine and alignment of the N-terminal segment of the P450DEHP with P450 4A1 indicated that the first four amino acids were absent. There were two amino acid differences between the two P450s in this 18-amino acid segment; in P450DEHP an alanine and a phenylalanine were substituted for serines in P450 4A1. The P450DEHP was found to catalyze the hydroxylation of several saturated fatty acids, having the highest turnover activity with laurate (82.1 nmol 12-OH-laurate formed/min/nmol P450). Myristate, palmitate, and stearate were also metabolized but at decreasing rates. Cytochrome b5 stimulated laurate 12-hydroxylation 10-fold in a reconstituted system. Laurate was not metabolized at its 11-carbon atom; however, the longer chain length fatty acids were metabolized at the (omega-1)-carbon atom in addition to the omega-carbon atom. A polyclonal antibody to the P450DEHP recognized three protein bands in liver microsomes from control and DEHP-treated rats on Western blot analysis, but only two protein bands from phenobarbital-treated rats.

Isolation and properties of lung 15-hydroxyprostaglandin dehydrogenase from pregnant rabbits.

A NAD-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was purified to a specific activity of over 25,000 nmol NADH formed/min/mg protein with 50 microM prostaglandin E1 as substrate from the lungs of 28-day-old pregnant rabbits. This represented a 2600-fold purification of the enzyme with a recovery of 6% of the starting enzyme activity. The lungs of pregnant rabbits were used because a 42- to 55-fold induction of the PGDH activity was observed after 20 days of gestation. The enzyme was purified by CM-cellulose, DEAE-cellulose, Sephadex G-75, octylamino-agarose, and hydroxylapatite chromatography. The enzyme could not be purified by affinity chromatography using NAD- or blue dextran-bound resins. The purified enzyme was specific for NAD and had a subunit molecular weight of 29,000. The optimal pH range for the oxidation of prostaglandin E1 was between 10.0 and 10.4 using 3-(cyclohexylamino)propanesulfonic acid as the buffer. The Km and Vmax values for prostaglandin E1 were 33 microM and 40,260 nmol/min/mg protein, respectively, while the Km and Vmax values for prostaglandin E2 were 59 microM and 43,319 nmol/min/mg protein, respectively. The Km for prostaglandin F2 alpha was four times the value for prostaglandin E1. The PGDH activity was inhibited by p-chloromercuriphenylsulfonic acid but the enzymatic activity was restored by the addition of dithiothreitol. n-Ethylmaleimide also produced a rapid decline in enzymatic activity but when NAD was included in the incubation system, no inhibition was observed.

Effect of phenobarbital treatment and cytochrome P-450 inhibitors on the laurate omega- and (omega - 1)-hydroxylase activities of rat liver microsomes.

The omega- and (omega - 1)-hydroxylase activities for lauric acid were investigated in rat liver microsomes. Treatment of rats with phenobarbital selectively induced the hydroxylation of the fatty acid (omega - 1)-hydroxylase activity two- to threefold, but had little effect on the omega-hydroxylation reaction. SKF 525-A, metyrapone, and alpha-naphthoflavone inhibited (omega - 1)-hydroxylation, but had only neglible effects on omega-hydroxylation. Metyrapone at 10(-4) inhibited the specific activity of (omega - 1)-hydroxylase 70% in phenobarbital-pretreated rats, but produced only a 10% inhibition of the omega-hydroxylation activity. alpha-Naphthoflavone at 10(-4)M inhibited (omega - 1)-hydroxylase activity 60% in untreated and beta-haphthoflavone-pretreated rats, while omega-hydroxylase activity was decreased only 20%. A selective effect was also observed when microsomes were stored overnight at 4 degrees C. Declines of 50% and 70% were observed in the (omega - 1)-hydroxylase activities after 24 and 48 hr, respectively, whereas omega-hydroxylation decreased only 10-20%. The differential effects on omega- and (omega - 1)-hydroxylase activities of a variety of conditions suggest that distinct cytochromes P-450 mediate the two fattty acid hydroxylases in liver microsomes.

Effects of diethyl phthalate and other plasticizers on laurate hydroxylation in rat liver microsomes.

Diethyl phthalate (DEP) is used in pharmaceutical coatings, cosmetics, and plastic films to wrap foods. There is a health concern associated with the exposure to certain phthalate esters because they belong to a class of compounds referred to as peroxisome proliferators which have been shown to increase the incidence of liver tumors when administered to rats. In this study, we have compared DEP to four other commonly used plasticizers, 2-diethylhexyl phthalate (DEHP), dibutyl phthalate (DBP), 2-diethylhezyl adipate (DEHA), and acetyltributyl citrate (ATBC), for their ability to induce the cytochrome P450-mediated fatty acid omega-hydroxylation system, which is one of the initial cellular responses when animals are treated with peroxisome proliferators. The administration of DEHP, DBP, and DEHA to rats increased the specific activity of laurate 12-hydroxylase from 2.8 +/- 1.1 in control rats to 30.3 +/- 11.6, 14.5 +/- 4.1, and 9.7 +/- 1.9 nmol 12-hydroxylaurate formed/min/nmol P450, respectively. In contrast, laurate 12-hydroxylase activity in DEP- and ATBC-treated rats were 4.4 +/- 1.2 and 4.4 +/- 1.0 nmol 12-hydroxylaurate formed/min/nmol P450, respectively. In addition, whereas DEHP increased peroxisomal palmitoyl-CoA oxidation 6-fold, DEP increased this activity only 1.3-fold. Two protein bands, at 51 and 52 kDa, were found to increase 6- to 12-fold in microsomes of DEHP-, DBP-, and DEHA-treated rats, but these bands were increased only 2-fold in DEP- or ATBC-treated rats.

Prostaglandin-metabolizing enzymes during pregnancy: characterization of NAD(+)-dependent prostaglandin dehydrogenase, carbonyl reductase, and cytochrome P450-dependent prostaglandin omega-hydroxylase.

Prostaglandins E2 and F2 alpha regulate a number of physiological functions in reproductive tissues, and concentrations of these bioactive modulators increase during pregnancy. Corresponding to the increase in circulating levels of prostaglandins during pregnancy is an increase in enzymes that metabolize these agents. Three prostaglandin-metabolizing enzymes induced during pregnancy are NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH), NADPH-dependent carbonyl reductase, and cytochrome P450-dependent prostaglandin omega- or 20-hydroxylase. This review discusses the biochemical properties, regulation, and possible functions of these three enzymes.

Characterization of cytochromes P450 in liver and kidney of rats treated with di-(2-ethylhexyl)phthalate.

A polyclonal antibody was made to a liver cytochrome P450 purified from di-(2-ethyl-hexyl)phthalate (DEHP)-treated Sprague-Dawley rats and was used to identify the CYP4A forms in liver and kidney cortex microsomes of control rats and rats treated with this peroxisome proliferator. Three clearly separated major protein bands were recognized on western blots in liver microsomes of control male rats or male rats treated with a single dose of DEHP, which, based on the description of relative mobility, tissue specificity, and sex dependent expression of CYP4A forms (Sundseth and Waxman (1992). J. Biol. Chem., 267, 801-810), correspond to the migration pattern of forms 4A1, 4A2, and 4A3 in clofibrate-treated rats. The administration of DEHP for 2 or 3 days caused a loss of resolution of two of the protein bands. The protein band corresponding to 4A2 was absent in liver or kidney cortex microsomes of DEHP-treated or control female rats and was not always visible in the livers of control male rats. The purified P450DEHP supported the hydroxylation of arachidonic acid at both the 19- and 20-carbon atoms with turnover rates of 1.4 +/- 0.2 and 22.7 +/- 2.5 nmoles per minute per nmol P450, respectively. No measurable amounts of hydroxylated products were obtained when prostaglandin E1, leukotriene B4, or testosterone were used as substrates. Another member of the CYP4 family, 4B1 from rabbit lung microsomes, was also recognized by this antibody on western blot analysis; however, rabbit lung form 4A4 showed only minimal cross-reactivity with this antibody.

Identification of 25,26-dihydroxyvitamin D3 as a rat renal 25-hydroxyvitamin D3 metabolite.

25,26-Dihydroxyvitamin D3 [25,26-(OH)2D3] was unequivocally identified as a major renal microsomal metabolite of 25-hydroxyvitamin D3 in rats fed a vitamin D sufficient diet. The structural assignment was based on a comparison of the high-performance liquid chromatograms of synthetic and in vitro generated 25,26-(OH)2D3 through four different systems, the ultraviolet absorbance and mass spectral characteristics of biological 25,26-(OH)2D3, and the chromatographic and mass spectral characteristics of the sodium metaperiodate cleavage product of the metabolite. The enzymic synthesis of 25,26-(OH)2D3 was inhibited 60--80% by a semipurified goat anti-rat NADPH--cytochrome P-450 reductase. This implicates cytochrome P-450 as the probable terminal oxidase of the 25-hydroxyvitamin D3-26-hydroxylase system. The methodology used to assay rat renal 25-OH-D3-hydroxylases is also discussed.

A prostaglandin omega-hydroxylase cytochrome P-450 (P-450PG-omega) purified from lungs of pregnant rabbits.

Cytochrome P-450-dependent prostaglandin omega-hydroxylation is induced over 100-fold during late gestation in rabbit pulmonary microsomes (Powell, W.S. (1978) J. Biol. Chem. 253, 6711-6716). Purification of cytochromes P-450 from lung microsomes of pregnant rabbits yielded three fractions. Two of these fractions correspond to rabbit lung P-450I (LM2) and P-450II (LM5), which together constitute 70-97% of total cytochrome P-450 in lung microsomes from nonpregnant rabbits. The third form, which we designate rabbit cytochrome P-450PG-omega, regioselectively hydroxylates prostaglandins at the omega-position in reconstituted systems with a turnover of 1-5 min-1. Titration with purified pig liver cytochrome b5, demonstrated a 4-fold maximum stimulation at a cytochrome b5 to a P-450 molar ratio of 1-2. Rabbit lung P-450PG-omega formed a typical type I binding spectrum upon the addition of prostaglandin E1 with a calculated K8 of 1 microM, which agreed reasonably well with the kinetically calculated Km of 3 microM. Cytochrome P-450PG-omega was isolated as a low-spin isozyme with a lambda max (450 nm) in the CO-difference spectrum distinguishable from P-450I (451 nm) and P-450II (449 nm). Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis demonstrated that although purified P-450PG-omega had a relatively low specific content (12.1 nmol mg-1), it appeared homogeneous with a calculated minimum Mr of 56,000, intermediate between rabbit LM4 and LM6. When lung microsomes from pregnant and nonpregnant rabbit were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a protein band, with a Mr identical to P-450PG-omega, was observed in the pregnant rabbit, whereas this band appeared to be very faint or absent in microsomes from the nonpregnant rabbit. Purification of cytochromes P-450 from nonpregnant rabbit lung yielded only P-450I and P-450II. P-450PG-omega appears to be a novel rabbit P-450, possessing high activity towards omega-hydroxylation of prostaglandins, and is greatly induced during pregnancy in rabbit lung.