Soluble epoxide hydrolase regulates hydrolysis of vasoactive epoxyeicosatrienoic acids.

The cytochrome P450-derived epoxyeicosatrienoic acids (EETs) have potent effects on renal vascular reactivity and tubular sodium and water transport; however, the role of these eicosanoids in the pathogenesis of hypertension is controversial. The current study examined the hydrolysis of the EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs) as a mechanism for regulation of EET activity and blood pressure. EET hydrolysis was increased 5- to 54-fold in renal cortical S9 fractions from the spontaneously hypertensive rat (SHR) relative to the normotensive Wistar-Kyoto (WKY) rat. This increase was most significant for the 14,15-EET regioisomer, and there was a clear preference for hydrolysis of 14, 15-EET over the 8,9- and 11,12-EETs. Increased EET hydrolysis was consistent with increased expression of soluble epoxide hydrolase (sEH) in the SHR renal microsomes and cytosol relative to the WKY samples. The urinary excretion of 14,15-DHET was 2.6-fold higher in the SHR than in the WKY rat, confirming increased EET hydrolysis in the SHR in vivo. Blood pressure was decreased 22+/-4 mm Hg (P:<0.01) 6 hours after treatment of SHRs with the selective sEH inhibitor N:, N:'-dicyclohexylurea; this treatment had no effect on blood pressure in the WKY rat. These studies identify sEH as a novel therapeutic target for control of blood pressure. The identification of a potent and selective inhibitor of EET hydrolysis will be invaluable in separating the vascular effects of the EET and DHET eicosanoids.

Soluble epoxide hydrolase in rat inflammatory cells is indistinguishable from soluble epoxide hydrolase in rat liver.

Soluble epoxide hydrolase (sEH) is a ubiquitous mammalian enzyme for which liver and kidney are reported to have the highest activity. We have shown that the soluble epoxide hydrolase (sEH) activity present in rat neutrophils and macrophages is kinetically, immunologically, and physically indistinguishable from rat liver cytosolic sEH. Cytosol from rat liver or inflammatory cells and recombinant rat sEH were incubated with trans-diphenylpropene oxide (tDPPO), a selective substrate for sEH. The tDPPO hydration activity we observed in inflammatory cell cytosol was lower than that from liver. The Km for tDPPO hydration observed in rat inflammatory cell cytosol was the same as the Km for rat liver cytosol (10 microM). Recombinant rat sEH and cytosol from rat liver or inflammatory cells were incubated with the sEH inhibitors, chalcone oxide, 4-fluorochalcone oxide, and 4-phenylchalcone oxide. The IC50 values were 40, 8, and 0.4 microM, respectively, in all samples. Furthermore, sEH activity could be completely immunoprecipitated out of the samples, and the amount of antibody required to do so was apparently identical, regardless of the source of enzyme. SDS-polyacrylamide gel electrophoresis followed by Western blot analysis revealed a single sEH band with a molecular weight of 62 kDa. Isoelectric focusing followed by Western blot analysis revealed multiple bands containing tDPPO-hydrating activity. Although the inflammatory cell bands had the same pattern as those from liver cytosol, the recombinant sEH showed a different banding pattern. These multiple bands were not an artifact of the IEF gel selected. Furthermore, in a 2-dimensional IEF gel, the bands re-migrated to the same position. The presence of sEH in inflammatory cells suggests that this enzyme may have an important endogenous function.

Inhibition of soluble and microsomal epoxide hydrolase by zinc and other metals.

Inhibition of xenobiotic-metabolizing enzymes by metals may represent an important mechanism in regulating enzyme activity. Fourteen cations were evaluated for inhibition of microsomal epoxide hydrolase (mEH) (mouse, rat, and human liver), soluble epoxide hydrolase (sEH) (mouse, rat, and human liver), and recombinant potato sEH. Of the metals tested, Hg2+ and Zn2+ were the strongest inhibitors of mEH, while Cd2+ and Cu2+ were also strong inhibitors of sEH (I50 for all approximately 20 microM). Nickel (divalent) and Pb2+ were moderate inhibitors, but Al2+, Ba2+, Ca2+, Co2+, Fe2+, Fe3+, Mg2+, and Mn2+ were weak inhibitors of both mEH and sEH (less than 50% inhibition by 1 mM metal). Six anions (acetate, bromide, chloride, nitrate, perchlorate, and sulfate) were tested and found to have no effect on the inhibition of sEH or mEH by cations. The kinetics and type of inhibition for zinc inhibition of sEH and mEH were examined for mouse, rat, human, and potato. Zinc inhibits mEH in a competitive manner. Inhibition of human and potato sEH was noncompetitive, but interestingly, zinc inhibition of mouse sEH was very strong and uncompetitive. Inhibition by zinc could be reversed by adding EDTA to the incubation buffer. Additionally, mouse liver microsomes and cytosol were incubated with these chelators. Following incubation at 4 degrees C, samples were dialyzed to remove chelator. Both mEH and sEH activity recovered was greater in samples treated with chelator than in control incubations. Similar treatment with the protease inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) did not affect enzyme activity recovered. During systemic inflammation, hepatic metallothionien is induced, and liver metal concentrations increase while serum metal concentrations are decreased. The inhibition of microsomal and soluble epoxide hydrolase by metals may represent a mechanism of down-regulation of enzyme activity during inflammation.

Identification of CYP2C9 as a human liver microsomal linoleic acid epoxygenase.

Leukotoxin (9,10-epoxy-12-octadecanoate) and isoleukotoxin (12, 13-epoxy-9-octadecenoate) are monoepoxides of linoleic acid, synthesized by a cytochrome P450 monooxygenase and possibly by an oxidative burst of inflammatory cells. Recent experiments in this laboratory have indicated that the toxicity of leukotoxin and isoleukotoxin is not due to these epoxides, but to the 9,10- and 12, 13-diol metabolites. Leukotoxin and isoleukotoxin are metabolized primarily by the soluble epoxide hydrolase to form leukotoxin diol. Investigations with recombinant cytochrome P450 enzymes have demonstrated that leukotoxin and isoleukotoxin can be formed by these enzymes. This study used a combination of experimental approaches to identify the major cytochrome P450 enzyme in human liver involved in linoleic acid epoxidation. The kinetic paramenters were determined; the K(m) of linoleic acid epoxidation by pooled human liver microsomes was 170 microM and the V(max) was 58 pmol/mg/min. Correlation analysis was performed using individual samples of human liver microsomes, and the best correlation of linoleic acid epoxidation activity was with tolbutamide hydroxylase activity, CYP2C9. Recombinant CYP2C9 was the most active in linoleic acid epoxygenation, and antibody and chemical inhibition also indicated the importance of CYP2C9. This enzyme, therefore, may serve as a therapeutic target in the treatment of inflammation in order to reduce the amount of circulating leukotoxin/isoleukotoxin and their related diols.

Inhibition of coumarin 7-hydroxylase activity in human liver microsomes.

Nine organic solvents and 47 commonly used P450 substrates and inhibitors were examined for their effects on coumarin 7-hydroxylase (CYP2A6) activity in human liver microsomes. Of the nine organic solvents examined (final concentration 1%, v/v), only methanol did not inhibit the 7-hydroxylation of coumarin (0.5 to 50 microM) by human liver microsomes. Dioxane and tetra-hydrofuran, which are structurally related to coumarin, were the most inhibitory solvents examined. Although the rates of coumarin 7-hydroxylation varied enormously among nine samples of human liver microsomes and cDNA-expressed CYP2A6 (Vmax = 179 to 2470 pmol/ mg protein/min), the Km for coumarin 7-hydroxylation was fairly constant (ranging from 0.50 to 0.70 microM). The following chemicals caused little or no inhibition of CYP2A6 as defined by a Ki > 200 microM: caffeine, chlorzoxazone, cimetidine, dextromethorphan, diazepam, diclofenac, erythromycin, ethinylestradiol, ethynyltestosterone, fluconazole, furafylline, furfural, hexobarbital, itraconazole, mephenytoin, methimazole, metronidazole, naringenin, naringin, nifedipine, norfloxacin, norgestrel, orphenadrine, quinidine, papaverine, phenacetin, pyrimethamine, ranitidine, spironolactone, sulfaphenazole, sulfinpyrazone, testosterone, tolbutamide, troleandomycin, and warfarin. In other words, these chemicals, at a final concentration of 100 microM, failed to inhibit CYP2A6 when the concentration of coumarin was equal to Km (0.50 microM). The following chemicals were classified as strong inhibitors of CYP2A6 (defined by Ki < 200 microM): clotrimazole, diethyldithiocarbamate, ellipticine, ketoconazole, 8-methoxypsoralen, 4-methylpyrazole, metyrapone, miconazole, alpha-naphthoflavone, nicotine, p-nitrophenol, and tranylcypromine. The potency with which each chemical inhibited the 7-hydroxylation of coumarin was independent of which sample of human liver microsomes was studied. One of the most potent inhibitors of coumarin 7-hydroxylase was 8-methoxypsoralen (methoxsalen), which was determined to be a mechanism-based inhibitor (suicide substrate) of CYP2A6 (k(inactivation) 0.5 min-1). With the exception of 8-methoxypsoralen, preincubation of human liver microsomes and NADPH with the aforementioned inhibitors did not increase their ability to inhibit CYP2A6. The most potent competitive inhibitor of CYP2A6 was tranylcypromine (Ki = 0.04 microM). Several of the chemicals that strongly inhibited CYP2A6, such as ketoconazole and tranylcypromine, are often used with the intention of selectively inhibiting human P450 enzymes other than CYP2A6. The results of this study underscore the need for a systematic evaluation of the specificity of commonly used P450 inhibitors.

Enhanced presynaptic facilitation of vascular adrenergic neurotransmission in spontaneously hypertensive rats.

1. The interaction between the vascular renin-angiotensin system and presynaptic beta-adrenoreceptors was examined in male and female normotensive and spontaneously hypertensive rats, using the in vitro perfused mesentery preparation. 2. Enhancement of the pressor response to periarterial nerve stimulation by isoprenaline was shown to be significantly greater in preparations from male and female spontaneously hypertensive rats compared to corresponding preparations from normotensive Wistar rats. 3. In preparations from normotensive and hypertensive animals, the potentiating effect of isoprenaline was prevented by pretreatment with propranolol or ICI 118 551, but not atenolol, implicating a beta 2-adrenoreceptor. 4. Angiotensin II enhanced the responses to peripheral nerve stimulation in preparation from normotensive and hypertensive animals. Enhancement was significantly greater in preparations from hypertensive animals. 5. The potentiation caused by isoprenaline was blocked by the angiotensin II receptor antagonist [Sar1-Ile8] angiotensin II, and by captopril. The potentiation following angiotensin II was unaffected by ICI 118 551. 6. These results suggest that stimulation of presynaptic beta 2-adrenoreceptors activates a localized angiotensin II system. No significant differences in this facilitatory system were observed between male and female animals, but the potentiation caused by activation of this system was considerably greater in the spontaneously hypertensive rats.

Development of a non-high pressure liquid chromatography assay to determine testosterone hydroxylase (CYP3A) activity in human liver microsomes.

The major pathway of testosterone oxidation by human liver microsomes is the formation of 6beta-hydroxytestosterone, which is catalyzed by CYP3A4/5 and which accounts for 75-80% of all metabolites formed. In the present study, we describe a non-high pressure liquid chromatography assay (HPLC) of CYP3A4/5 activity based on the release of tritium (with formation of tritiated water) upon incubation of [1,2,6,7-3H]testosterone with human liver microsomes and NADPH. Unreacted testosterone and its metabolites were quantitatively extracted from the incubation mixture with activated charcoal under conditions that resulted in no extraction of tritiated water. The amount of tritiated water formed was quantified by liquid scintillation spectrometry and compared with the amount of hydroxylated testosterone metabolites formed, as determined by HPLC. Rates of tritium release from [1,2,6, 7-3H]testosterone paralleled rates of testosterone 6beta-hydroxylation as a function of incubation time, the amount of microsomal protein, and the concentration of substrate (which yielded identical apparent Km and Vmax values). The sample-to-sample variation in tritium release from [1,2,6,7-3H]testosterone with a panel of human liver microsomes was highly correlated with rates of testosterone 6beta-hydroxylation and terfenadine metabolism, two commonly used markers of CYP3A activity. Several recombinant human P450 enzymes were incubated with [1,2,6,7-3H]testosterone, and only cDNA-expressed CYP3A4 catalyzed a high rate of tritium release. The close agreement between the tritium-release assay and HPLC procedure for measuring testosterone oxidation indicates that tritium release from [1,2,6,7-3H]testosterone provides a simple and rapid alternative to the HPLC procedure for measuring CYP3A4/5 activity in human liver microsomes. However, the tritium-release assay may have limited value in measuring CYP3A activity in liver microsomes from other species due to the presence of other P450 enzymes that can catalyze tritium release from [1,2,6,7-3H]testosterone.

Development of a non-high pressure liquid chromatography assay to determine [14C]chlorzoxazone 6-hydroxylase (CYP2E1) activity in human liver microsomes.

The activity of liver microsomal CYP2E1 is commonly measured as the rate of 5-chloro-2-benzoxazolone (chlorzoxazone) 6-hydroxylation, which requires separation of 6-hydroxychlorzoxazone and chlorzoxazone by high pressure liquid chromatography (HPLC). In the present study, we describe a solvent extraction (non-HPLC) assay for measuring CYP2E1 activity, based on the 6-hydroxylation of [14C]chlorzoxazone. When [14C]chlorzoxazone was incubated with human or rat liver microsomes in the presence of NADPH, the major product formed was 6-[14C]hydroxychlorzoxazone. Unreacted [14C]chlorzoxazone was quantitatively extracted from the incubation mixture with dichloromethane under conditions that resulted in approximately 45% extraction of 6-[14C]hydroxychlorzoxazone. The amount of 6-[14C]hydroxychlorzoxazone remaining in the aqueous incubation mixture ( approximately 55% of the total amount formed) was quantified by liquid scintillation spectrometry. The limit of detection for this assay was 100 pmol of 6-[14C]hydroxychlorzoxazone. The solvent extraction procedure was validated by comparing the rates of formation of 6-[14C]hydroxychlorzoxazone with those determined by HPLC under a variety of experimental conditions. The close correspondence between the two analytical methods suggests that the extraction procedure for measuring 6-[14C]hydroxychlorzoxazone provides a simple, sensitive, and rapid alternative to the HPLC procedure for measuring CYP2E1 activity. In rats, the assay is not specific for CYP2E1 because CYP1A1 also catalyzes the 6-hydroxylation of chlorzoxazone. Recombinant human CYP1A1 also catalyzed the 6-hydroxylation of chlorzoxazone (at (1)/(5) the rate of CYP2E1), although CYP1A1 is not expressed in human liver microsomes. The non-HPLC assay was used to investigate the postulated role of CYP1A2 in the 6-hydroxylation of chlorzoxazone by human liver microsomes. Recombinant CYP1A2 did not catalyze the 6-hydroxylation of chlorzoxazone, and studies with 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline, which inhibits CYP1A2 but not CYP2E1, indicated that, in human liver microsomes, the 6-hydroxylation of chlorzoxazone is catalyzed by CYP2E1 with little or no contribution from CYP1A2 enzymes over a wide range of substrate concentrations.

The chronic effects of beta-adrenoreceptor antagonists on the efflux of tritiated noradrenaline from sympathetic nerves of the pithed rat.

The effect of chronic administration of two beta-adrenoreceptor blocking drugs, propranolol and timolol, on transmitter noradrenaline release from sympathetic nerves has been investigated in vivo using the pithed rat preparation. Oral treatment for 4 weeks with either propranolol (46.3 mg/kg/day) or timolol (7.1 mg/kg/day) significantly raised the stimulation frequency threshold for release of radioactivity on stimulation of the whole spinal outflow of the pithed rat after i.v. injection of 3H-NA. No differences in the stimulation-evoked rise in mean arterial pressure were observed between control or treated rats nor was the heart rate response to stimulation altered after timolol treatment. However, in propranolol treated rats the mean rises in heart rate were significantly higher with 3 and 30 Hz stimulation than in control rats. Timolol treatment significantly increased the blood concentration and lowered the heart content of 3H-NA whilst propranolol treatment did not significantly change either blood or heart levels. Log dose-response curves for mean rises in heart rate after i.v. isoprenaline were not shifted to the right in either propranolol or timolol treated pithed rats. With the lowest doses of isoprenaline (1 and 25 ng), the mean rises in heart rate in timolol treated rats were significantly greater than in the controls. Thus chronic administration of propranolol or timolol decreased the stimulation-induced increase in plasma 3H-NA but this change in release was not related to reduced rises in blood pressure or heart rate.

Effects of chronic administration of atenolol on the in situ perfused mesentery of normotensive and hypertensive rats.

1. Atenolol 50 mg kg-1 was administered daily for 7 to 21 days to male normotensive Wistar rats and to Okamoto spontaneously hypertensive rats. Blood pressure and heart rate were monitored daily. 2. Following atenolol treatment the response to exogenous noradrenaline and to periarterial nerve stimulation was measured in the in situ blood perfused mesentery. 3. In normotensive animals, treatment with atenolol for 7 days had little effect on the responses of the mesenteric vasculature to either exogenous noradrenaline or to periarterial nerve stimulation. After 21 days, the response to noradrenaline was little changed, but there was a statistically significant decrease in the response to periarterial nerve stimulation at the higher stimulation frequencies (16 and 35 Hz). 4. In spontaneously hypertensive animals, treatment with atenolol for 7 days did not significantly affect the response of the mesenteric vasculature to either noradrenaline or periarterial nerve stimulation; however, after treatment for 21 days, responses to exogenous noradrenaline were reduced, while responses to periarterial nerve stimulation were even more markedly reduced, particularly at the lower stimulus frequencies. 5. The time course of the observed effects suggests that, over time, atenolol induces changes other than the simple blockade of beta-adrenoceptors which can be demonstrated to be present immediately. The decreased responses to periarterial nerve stimulation in normotensive and hypertensive animals suggest that the changes induced affect presynaptic mechanisms of noradrenaline release (either directly or indirectly), rather than changes in postsynaptic receptor sensitivity or vascular reactivity. The observation that the changes are more evident in hypertensive animals indicates that they may play an important role in the antihypertensive action of atenolol.

Response of the rat mesenteric vasculature to chronic treatment with nitrendipine alone and in combination with atenolol: evidence of a significant drug interaction.

1. Nitrendipine 3 mg kg-1 was administered alone and in combination with atenolol 50 mg mg-1 day for 21 days to male normotensive Wistar rats and to spontaneously hypertensive Japanese Okamoto rats. Blood pressure was monitored daily. 2. Systolic blood pressure was decreased in normotensive Wistars and SHRs by nitrendipine alone and in combination. In both cases the decrease was greater in the hypertensive animal. There was no evidence of the combination having an additive hypotensive effect. 3. Following treatment, the response to exogenous noradrenaline (NA) and periarterial nerve stimulation (PNS) was measured in the in situ blood perfused mesentery. 4. Treatment with nitrendipine and the combination reduced the response to exogenous noradrenaline; with both, the reduction was greater in the hypertensive animal. The combination produced a larger reduction in response than nitrendipine alone. 5. Treatment with nitrendipine alone reduced the response to PNS in both normotensive and hypertensive animals, although this effect was greater in the SHR. 6. Combination treatment failed to change the response to electrical stimulation in the SHR, while in the normotensive rat it resulted in a large increase in response to higher frequency (16 and 35 Hz) stimulation. 7. As nitrendipine given alone reduced the response to PNS, and as we have previously shown a similar effect with atenolol given alone (Draper, Kingsbury, Redfern & Todd, 1992), the effect of the combination of nitrendipine and atenolol on responses to PNS is apparently influenced by some interaction between the two drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

The chronic effects of beta-adrenoreceptor blocking agents on transmitter overflow in the anococcygeus muscle of the rat.

1 The effect of chronic administration of three beta-adrenoreceptor blocking agents, propranolol, timolol and atenolol on transmitter release from sympathetic nerves has been investigated in the isolated anococcygeus muscle of the rat. 2 Oral treatment for 11 weeks with either propranolol (12 mg/kg/day), or timolol (1.2 mg/kg/day) but not atenolol (12 mg/kg/day) significantly increased the stimulation-induced overflow of radioactivity following preincubation of the tissue with 3H-NA. 3 No increase in end-organ sensitivity, as measured in terms of responsiveness to exogenous acetylcholine, tyramine or NA, was observed. 4 The ineffectiveness of atenolol compared to the other beta-adrenoreceptor blocking drugs may be related to the finding that, in acute experiments, atenolol inhibited tissue uptake of 3H-NA. 5 It is postulated that increased release of transmitter may be related to prolonged blockade of presynaptic beta-receptors and a resultant increase in presynaptic receptor sensitivity.

Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity.

The stability of cytochrome P450 enzymes, cytochrome b5, and NADPH-cytochrome c reductase was examined in (A) human liver samples frozen in liquid nitrogen and stored at -80 degrees C, (B) human liver microsomes suspended in 250 mM sucrose and stored at -80 degrees C, and (C) human liver microsomes subjected to as many as 10 cycles of thawing and freezing. In study A, microsomes from five human livers were prepared from fresh (unfrozen) tissue and from tissue that was stored frozen at -80 degrees C for 1, 2, 4, or 6 months. The apparent concentration of cytochromes P450 and b5 and the activity of NADPH-cytochrome c reductase decreased 20-40% as a result of freezing the liver, regardless of whether the liver was stored for 1 or 6 months. Similar decreases were observed in the activities of cytochrome P450 enzymes belonging to several gene families, namely CYP1A2 (7-ethoxyresorufin O-dealkylation and caffeine N3-demethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (tolbutamide methylhydroxylation), CYP2C19 (S-mephenytoin 4'- hydroxylation), CYP2D6 (dextromethorphan O-de-methylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4solidus5 (testosterone 6beta-hydroxylation), and CYP4A9solidus11 (lauric acid 12-hydroxylation). Freezing human liver did not convert cytochrome P450 to its inactive form, cytochrome P420, but it increased the contamination of liver microsomes with hemoglobin or other heme-containing proteins, which resulted in a uniform decrease in the specific activity of cytochromes P450 and b5 and in the specific activity of all P450 enzymes. In study B, the concentration of cytochromes P450 and b5, the activity of NADPH-cytochrome c reductase, and the activity of individual cytochrome P450 enzymes were determined in 10 samples of human liver microsomes stored at -80 degrees C for approximately 0, 1, or 2 years. The sample-to-sample variation in the concentration and activity of cytochrome P450, cytochrome b5, and NADPH-cytochrome c reductase was nominally affected by long-term storage of human liver microsomes at -80 degrees C, indicating there was no differential loss of cytochrome P450 activity, cytochrome b5 concentration, or NADPH-cytochrome c reductase activity. In study C, microsomes from a pool of human livers were subjected to 1, 2, 3, 5, 7, or 10 cycles of freezing at -80 degrees C followed by thawing at room temperature. Freezing/thawing liver microsomes for up to 10 cycles did not convert cytochrome P450 to P420, nor did it cause significant loss of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5, or CYP4A9/11 activity. Overall, these results suggest that our current methods for storing and processing human liver are well suited to preserving microsomal P450 enzyme activity.