ABSTRACT:Lipophilic bile acids are suggested to be involved in the endogenous expression of CYP3A4 in human and experimental animals as ligands of nuclear receptors. To verify the nuclear receptor specificity, the bile acid-mediated induction of CYP3A4 has been studied in vitro and in vivo in the present study. Lithocholic acid (LCA) strongly enhanced the activities of the CYP3A4 reporter gene, which contained multiple nuclear receptor binding elements, in both HepG2 and LS174T cells. The introduction of small interfering RNA for human vitamin D receptor (VDR), but not for human pregnane X receptor, reduced the LCA-induced activation of the reporter gene in these cells, suggesting the major role of VDR in the LCA induction of CYP3A4. Consistently, oral administration of LCA (100 mg/kg/day for 3 days) increased Cyp3a protein levels in the intestine but not in the liver, where a negligible level of VDR mRNA is detected. The selective role of VDR was tested in mice with the adenoviral overexpression of the receptor. Oral administration of LCA had no clear influence on the CYP3A4 reporter activity in the liver of control mice. In mice with the adenovirally expressed VDR, LCA treatment (100 or 400 mg/kg/day for 3 days) resulted in the enhanced reporter activities and increased levels of Cyp3a proteins in the liver. These results indicate the selective involvement of VDR, but not pregnane X receptor, in the LCA-mediated induction of both human and mouse CYP3As in vivo.

Sterol regulatory element-binding protein-2 (SREBP-2) is a key transcription factor for the cholesterol homeostasis. Recent studies have suggested the association of CYP3A enzymes, major drug-metabolizing enzymes, with cholesterol metabolism. In the present study, we have investigated a possible involvement of SREBP-2 in hepatic Cyp3a11 expression. Feeding a low-cholesterol diet (LCD) to mice activated hepatic SREBP-2 whereas it attenuated hepatic Cyp3a11 expression. These phenomena were reversed by cholesterol supplementation to LCD. In reporter assays, the overexpression of constitutively active SREBP-2 reduced Cyp3a11 reporter activity through the region from Ϫ1581 to Ϫ1570 of Cyp3a11. This region contained a putative hepatocyte nuclear factor-4␣ (HNF-4␣) binding motif, and HNF-4␣, but not SREBP-2, bound to the motif in in vitro binding assays. With the mutation or deletion of this motif, the SREBP-2-dependent suppression of Cyp3a11 expression disappeared in reporter assays. In pull-down assays and coimmunoprecipitation assays, SREBP-2 bound to peroxisome proliferator-activated receptor ␥ coactivator-1␣ (PGC-1␣), a major coactivator for HNF-4␣, via its transactivation domain and inhibited the interaction between HNF-4␣ and PGC-1␣ in vitro. A mutant SREBP-2 lacking the transactivation domain consistently failed to reduce Cyp3a11 reporter activity. Furthermore, PGC-1␣ overexpression relieved the SREBP-2-mediated reduction of Cyp3a11 reporter activity. Finally, chromatin immunoprecipitation assays demonstrated that the extent of PGC-1␣ binding to the Cyp3a11 promoter was reduced by LCD-feeding in mouse livers. In conclusion, activated SREBP-2 interacts with PGC-1␣ in mouse livers at reduced cholesterol intake. This results in the reduced PGC-1␣ recruitment to HNF-4␣ on the Cyp3a11 promoter and the subsequent downregulation of Cyp3a11 expression.

The sensitivity of the gastrointestinal tract to stress has been demonstrated in clinical settings for over half a century.1) Psychological and physical stress stimuli are associated with gastrointestinal dysfunction, including abdominal pain and diarrhea. [2][3][4] One clinical study demonstrated stress-induced alteration of colonic motility in both healthy subjects and patients with irritable bowel syndrome.5) The effects of stress on the gastrointestinal motility are conflicting: an acceleration as well as a delay of the motility by stress have been reported in small intestine, [6][7][8][9] and in colon. 7,[10][11][12][13] However, the etiology of intestinal dysfunction due to stress has not been completely clarified, and the lack of an appropriate animal model has been an obstacle to studies on the mechanism. We developed an experimental system with which to evaluate the stress effect on small intestinal motility, 14) and have since improved the system. 15) Using this improved method, we observed that small intestinal motility was inhibited by restraint stress. 15) Since it is well known that the sympathetic nervous system is strongly stimulated by acute stress, 16,17) we examined the influence of the adrenoceptor antagonists, prazosin (a 1 -antagonist), yohimbine (a 2 -antagonist), propranolol (b 1 /b 2 -antagonist), atenolol (b 1 -antagonist), ICI-118551 (b 2 -antagonist) and SR59230A (b 3 -antagonist) in order to clarify the type of adrenoceptors involved in the inhibition of small intestinal motility by restraint stress. MATERIALS AND METHODS AnimalsMale Wistar rats (6-weeks old, 150-200 g) were purchased from Japan Shizuoka Laboratory Animal Center (Hamamatsu, Japan), and were used in all the experiments according to the Guideline for Animal Experimentation of Tohoku Pharmaceutical University. The animals were housed in a wire-mesh cage [26 (W)ϫ38 (D)ϫ19 (H) cm] placed in a chamber with a constant temperature (23Ϯ1°C), humidity (55Ϯ5%) and a 12 h light-dark cycle (light 8:30 a.m. to 8:30 p.m.). The animals were given free access to tap water and rat chow.Reagents Sodium chromate ( 51 Cr) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ, U.S.A.), heparin sodium from Novo Nordisk (Bagsvaerd, Denmark), pentobarbital sodium (Nembutal ® , 50 mg/ml) from Dainippon Pharmaceutical (Osaka, Japan), yohimbine HCl and DL-propranolol HCl from Nacalai Tesque (Kyoto, Japan), prazosin HCl from Sigma Aldrich Japan (Tokyo), atenolol from Wako Pure Chemicals (Tokyo), and ICI-118,551 Nawas a kind gift of Mitsubishi-Tokyo Pharmaceuticals (Tokyo). All other chemicals were of reagent grade.Cannula Implantation Cannula implantation was performed according to the method of Tsukada et al.15) The rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.), the abdomen opened by a midline incision (approximately 2-3 cm length), and a chronic indwelling silicon cannula (0.5 mm internal diameter and 0.9 mm outer diameter, medical tube SH No. 00; Kaneka, Tokyo) was inserted into the duodenum toward the jejunum and ...

We previously reported that small intestinal motility was significantly inhibited by restraint stress, but not by footshock stress. In the present study, we found that plasma b b-endorphin levels were more significantly elevated by footshock stress than restraint stress, and that preloading of footshock stimulus canceled the inhibition of small intestinal motility by restraint stress. Pretreatment with the m m-opioid receptor antagonist naltrexone significantly attenuated this canceling effect of footshock stimulus. These results suggest that footshock stimulus may cancel the inhibition of small intestinal motility by restraint stress via activation of m m-opioid receptors.

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