Renal microsomal cytochrome P-450 monooxygenasedependent metabolism of arachidonic acid generates a series of regioisomeric epoxyeicosatrienoic acids that can be further metabolized by soluble epoxide hydrolase to the corresponding dihydroxyeicosatrienoic acids. Evidence exists that these metabolites affect renal function and, in particular, blood pressure regulation. To examine this possibility, blood pressure and renal arachidonic acid metabolism were examined in mice with a targeted disruption of the soluble epoxide hydrolase gene. Systolic blood pressure of male soluble epoxide hydrolase-null mice was lower compared with wild-type mice in both the absence and presence of dietary salt loading. Both female soluble epoxide hydrolase-null and wild-type female mice also had significantly lower systolic blood pressure than male wild-type mice. Renal formation of epoxyeicosatrienoic and dihydroxyeicosatrienoic acids was markedly lower for soluble epoxide hydrolase-null versus wild-type mice of both sexes. Although disruption of soluble epoxide hydrolase in female mice had minimal effects on blood pressure, deletion of this gene feminized male mice by lowering systolic blood pressure and altering arachidonic acid metabolism. These data provide the first direct evidence for a role for soluble epoxide hydrolase in blood pressure regulation and identify this enzyme as a novel and attractive target for therapeutic intervention in hypertension. Mammalian soluble epoxide hydrolase (sEH)1 is a cytosolic enzyme expressed in multiple tissues that catalyzes the conversion of a diverse group of epoxides to their corresponding diols (1, 2). The broad spectrum of xenobiotic epoxides metabolized by this enzyme suggests a role in the protection of cells against the potentially harmful effects of these compounds (3). However, sEH hydrates fatty acid epoxides most effectively, with epoxides of arachidonic acid (AA) being among the best substrates (4 -6). This metabolism may result in the formation of metabolites with greater or less biological activity, indicating that sEH may have important physiological functions. Intracellular metabolism of AA by prostaglandin H2 synthase, lipoxygenases, and the cytochrome P-450 (CYP) monooxygenase system generates a number of metabolites, collectively termed eicosanoids, with important biological and cell signaling roles (7-12). CYP-dependent metabolism of AA (Fig. 1) generates three primary classes of metabolites: mid-chain cis-trans-conjugated dienols (5-, 8-, 9-, 11-, 12-and 15-hydroxyeicosatetraenoic acids (HETEs)); -terminal alcohols (16-through 20-hydroxyeicosatetraenoic acids); and, cis-epoxyeicosatrienoic acids (6-, 8,9-, 11,12-, and 14,15 epoxyeicosatrienoic acids (EETs)) (13). EETs have been shown in vitro to be efficiently hydrated to their corresponding dihydroxyeicosatrienoic acids (DHETs) by sEH (5).Evidence exists for a variety of renal functions attributable to EETs and DHETs generated by CYP-dependent epoxygenase activity and sEH, respectively. Most notably, evidence exists for ...

The biological action of adrenomedullin, a novel hypotensive peptide, on bovine aortic endothelial cells, was examined. The specific binding of adrenomedullin to these cells was observed, and adrenomedullin was found to induce intracellular cAMP accumulation in a dose-dependent manner. EC50 for the cAMP accumulation was about 100 times lower than the apparent IC50 for the binding assay. Adrenomedullin also induced increase of intracellular free Ca2+ in endothelial cells in a dose-dependent manner. The Ca2+ response to adrenomedullin was biphasic with an initial transient increase due to the release from thapsigargin-sensitive intracellular Ca2+ storage and a prolonged increase by influx through the ion channel on the plasma membrane. This intracellular free Ca2+ increase resulted from phospholipase C activation and inositol 1,4,5-trisphosphate formation, and seemed to cause nitric oxide synthase activation by monitoring intracellular cGMP accumulation. Both cAMP accumulation and Ca2+ increased responses to adrenomedullin were mediated by cholera toxin-sensitive G protein, but the two signal transduction pathways were independent. Thus, the results suggest that adrenomedullin elicits the hypotensive effect through at least two mechanisms, a direct action on vascular smooth muscle cells to increase intracellular cAMP and an action on endothelial cells to stimulate nitric oxide release, with both leading to vascular relaxation.

Supplement of 1% lithocholic acid (LCA) in the diet for 5-9 days resulted in elevated levels of the marker for liver damage aspartate aminotransferase and alkaline phosphatase activities in both farnesoid X receptor (FXR)-null and wild-type female mice. The levels were clearly higher in wild-type mice than in FXR-null mice, despite the diminished expression of a bile salt export pump in the latter. Consistent with liver toxicity marker activities, serum and liver levels of bile acids, particularly LCA and taurolithocholic acid, were clearly higher in wild-type mice than in FXR-null mice after 1% LCA supplement. Marked increases in hepatic sulfating activity for LCA (5.5-fold) and hydroxysteroid sulfotransferase (St) 2a (5.8-fold) were detected in liver of FXR-null mice. A 7.4-fold higher 3␣-sulfated bile acid concentration was observed in bile of FXR-null mice fed an LCA diet compared with that of wild-type mice. Liver St2a content was inversely correlated with levels of alkaline phosphatase. In contrast, microsomal LCA 6␤-hydroxylation was not increased and was in fact lower in FXRnull mice compared in wild-type mice. Clear decreases in mRNA encoding sodium taurocholate cotransporting polypeptide, organic anion transporting polypeptide 1, and liver-specific organic anion transporter-1 function in bile acid import were detected in LCA-fed mice. These transporter levels are higher in FXR-null mice than wild-type mice after 1% LCA supplement. No obvious changes were detected in the Mrp2, Mrp3, and Mrp4 mRNAs. These results indicate hydroxysteroid sulfotransferase-mediated LCA sulfation as a major pathway for protection against LCA-induced liver damage. Furthermore, Northern blot analysis using FXR-null, pregnane X receptor-null, and FXR-pregnane X receptor double-null mice suggests a repressive role of these nuclear receptors on basal St2a expression.

-This review showed the common pathogenic mechanism in the development of non-alcoholic or alcoholic steatohepatitis. In particular, we describe the role of innate immune system and oxidative stress caused by gut-derived endotoxin. Gut-derived endotoxin plays an important role in alcoholic liver injury. It was reported that acute ethanol administration reduced activation of Kupffer cells. It is therefore possible that alcohol-induced hepatocellular damage occurs as a result of bacterial or endotoxin translocation under a reduction of the reticuloendothelial system (RES) function in alcoholic liver disease (ALD). On the other hand, recently, attention has been directed toward the effect of ethanol ingestion on Kupffer cell function, which is stimulated by gut-derived endotoxin via mechanisms dependent on increased gut permeability and the possible relationship between Kupffer cells and alcohol-induced liver injury. It is generally accepted that activation of the innate immune system and increased release of proinflammatory cytokines and other mediators plays an important role in the development of ALD. It was shown that Kupffer cells activation by endotoxin via Toll-like receptor (TLR-4) is involved in alcoholinduced liver injury and that ethanol-induced oxidative stress is important in the regulation of transcription factor NF-κB activation and that cytokine production by Kupffer cells. TNF-α and free radicals are produced in early alcohol-induced liver injury. In support of this finding, the pathology caused by alcohol was blocked nearly completely in TNF-α receptor 1. Many pathways have been suggested to contribute to the ability of ethanol to induce a state of oxidative stress. One central pathway appears to be the induction of the CYP2E1 form of cytochrome P450 enzymes by ethanol. Initial efforts to clarify the mechanisms that promote the progression from steatosis to steatohepatitis somewhat artificially divides disease mechanisms into "first and second" hit. The best candidates for these second hits were considered to be oxidative stress (CYP2E1 induction) and associated lipid peroxidation and cyokines, principally, TNF-α. Some of the most definitive data on the importance of the innate immune system or oxidative stress in the pathogenesis of liver disease come from studies of alcoholic and non-alcoholic steatohepatitis in animals.

Cytosolic sulfotransferase catalyzes sulfoconjugation of relatively small lipophilic endobiotics and xenobiotics. At least 44 cytosolic sulfotransferases have been identified from mammals, and based on their amino acid sequences, these forms are shown to constitute five different families. In humans, 10 sulfotransferase genes have been identified and shown to localize on at least five different chromosomes. The enzymatic properties characterized in the recombinant forms indicate the association of their substrate specificity with metabolisms of such nonpeptide hormones as estrogen, corticoid, and thyroxine, although most forms are also active on the sulfation of various xenobiotics. Genetic polymorphisms are observed on such human sulfotransferases as ST1A2, ST1A3, and ST2A3.

Hepatocyte nuclear factor 4alpha (HNF4alpha) is an important transcription factor in hepatic gene expression. Here, we have investigated the role of HNF4alpha in the expression of drug-metabolizing enzymes and transporters in human hepatocytes using an adenovirus expressing human HNF4alpha-small interfering RNA (hHNF4alpha-siRNA). The hHNF4alpha-siRNA effectively reduced the mRNA and nuclear protein levels of hHNF4alpha in a concentration-dependent manner. The hHNF4alpha-siRNA also decreased the mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A1, UGT1A9, SULT2A1, ABCB1, ABCB11, ABCC2, OATP1B1 and OCT1, as well as those of PXR and CAR. To discern the role of these nuclear receptors, we co-infected hepatocytes with hHNF4alpha-siRNA and PXR- or CAR-expressing adenovirus. The hHNF4alpha-siRNA-induced reductions of the enzyme and transporter mRNA levels were not restored except CYP2B6 mRNA levels, which were returned to the control level by overexpressing CAR. Furthermore, although hHNF4alpha-siRNA did not significantly affect the fold-induction of CYP2B6, CYP2C8, CYP2C9, or CYP3A4 mRNA levels following treatment with CYP inducers, the levels in hHNF4alpha-suppressed cells fell significantly compared to the control. These results suggest that HNF4alpha plays a dominant role in the expression of drug-metabolizing enzymes and transporters in human hepatocytes, and that HNF4alpha expression levels is a possible determinant for inter-individual variations in the expression of these enzymes and transporters.

Growing evidence indicates that the innate immune system and oxidative stress caused by gut-derived endotoxins play a key role in alcoholic liver disease (ALD). Intracellular mechanisms associated with endotoxin-induced signaling play a crucial role in the initiation and progression of ALD. It is now widely accepted that activation of the innate immune system and increased release of pro-inflammatory cytokines and other mediators play an important role in the development of ALD. Accumulating evidence suggests that alcohol-mediated upregulation of CYP2E1 expression may initiate lipid peroxidation via reactive oxygen species. Non-alcoholic steatohepatitis (NASH) is a liver disease characterized by histopathological features similar to those observed in ALD, but in the absence of significant alcohol consumption. Initial efforts to clarify the mechanisms that promote the progression from steatosis to steatohepatitis somewhat artificially divided disease mechanisms into "first and second hits." This model considered the development of steatosis to be the "first hit," increasing the sensitivity of the liver to the putative "second hit," leading to hepatocyte injury, inflammation, and oxidative stress. We have emphasized the important role of gut-derived bacterial toxins, the innate immune system, and oxidative stress in the common pathogenic mechanism in ALD and NASH progression.

ABSTRACT:Orally administered astemizole is well absorbed but undergoes an extensive first-pass metabolism to O-desmethylastemizole. Desmethylastemizole is formed in the human microsomal systems of the small intestine as well as the liver, which suggests the role of cytochromes P450 (P450s) in the first-pass metabolism of astemizole. Human P450s involved in the O-demethylation of astemizole have, however, not been identified, and the involvement of twelve known drug-metabolizing P450s were denied. During the course of the P450 identification study, higher activities of the astemizole O-demethylation in the rabbit small intestine than in the liver (about 3-fold) were found. These data suggest the possible involvement of CYP2J, since P450 included in this subfamily is dominantly expressed in the small intestine of rabbits. Therefore, CYP2J2 cDNA