Zoxazolamine-induced stimulation of cardiomyogenesis from embryonic stem cells is mediated by Ca2+, nitric oxide and ATP release.

Ca2+-activated potassium (KCa) channels of small and intermediate conductance influence proliferation, apoptosis, and cell metabolism. We analysed whether prolonged activation of KCa channels by zoxazolamine (ZOX) induces differentiation of mouse embryonic stem (ES) cells towards cardiomyocytes. ZOX treatment of ES cells dose-dependent increased the number and diameter of cardiac foci, the frequency of contractions as well as mRNA expression of the cardiac transcription factor Nkx-2.5, the cardiac markers cardiac troponin I (cTnI), α-myosin heavy chain (α-MHC), ventricular myosin light chain-2 (MLC2v), and the pacemaker hyperpolarization-activated, cyclic nucleotide-gated 4 channel (HCN4). ZOX induced hyperpolarization of membrane potential due to activation of IKCa, raised intracellular Ca2+ concentration ([Ca2+]i) and nitric oxide (NO) in a Ca2+-dependent manner. The Ca2+ response to ZOX was inhibited by chelation of Ca2+ with BAPTA-AM, release of Ca2+ from intracellular stores by thapsigargin and the phospholipase C (PLC) antagonist U73,122. Moreover, the ZOX-induced Ca2+ response was blunted by the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) as well as the specific P2Y1 antagonist MRS 2,179, suggesting purinergic receptor-stimulated signal transduction. Consequently, ZOX initiated ATP release from differentiating ES cells, which was inhibited by the chloride channel inhibitor NPPB and the gap junction inhibitor carbenoxolone (CBX). The stimulation of cardiomyogenesis by ZOX was blunted by the nitric oxide synthase (NOS) inhibitor l-NAME, as well as CBX and NPPB. In summary, our data suggest that ZOX enhances cardiomyogenesis of ES cells by ATP release presumably through gap junctional hemichannels, purinergic receptor activation and intracellular Ca2+ response, thus promoting NO generation.

Acute and Subchronic Toxicity Studies of Aqueous Extract of Desmodium adscendens (Sw) DC.

Extracts of Desmodium adscendens (Sw) DC are used for the treatment of various diseases but limited toxicological evaluations have been done on the medicinal plant. This study investigates toxicity effects of the leave extract of D adscendens, and the possibility of drug-drug interaction of the plant extract when co-administered with other drugs. Oral administrations of leaf extract of D adscendens to white Wistar rats in an acute toxicity studies allowed the estimation of an LD50 (median lethal dose) value of 1122 mg/kg body weight. In a subchronic toxicity studies, the plant extract caused a decrease in zoxazolamine paralysis time and prevented thiopentone from causing sleep in test animals compared to controls. Overall, the results are consistent with the plant extract being safe at the doses administered in humans. However, the induction of the CYP enzymes is an indication of a possible drug interaction when the plant extract is co-administered with other drugs.

Effect of intermittent hypobaric hypoxia on efficacy & clearance of drugs.

BACKGROUND & OBJECTIVES: People travelling to high altitude for occupational, recreational or religious purposes are mostly healthy and fit but sometimes they use drugs for common ailments like influenza, acute mountain sickness or chronic disease like diabetes. Limitation of oxygen at high altitude may compromise metabolism of drugs. Hence, we undertook this study to assess the effect of hypobaric hypoxia on some commonly used drugs in rats and rabbits. METHODS: Effect of intermittent hypobaric hypoxia on phenotypic expression of anesthetic drugs pentabarbitone, thiopentone and zoxazolamine (sleeping time) was assessed in rats exposed to 282.4 mm Hg equivalent to 25000 feet in a decompression chamber. Plasma clearance of some commonly used drugs was investigated in rabbits exposed to 429 mm Hg equivalent to 15000 feet. Pharmacokinetic parameters were computed by plotting drug concentration versus time curve on semi log scale. RESULTS: A significant delay in regaining rightening reflex was observed in rats exposed to intermittent hypobaric hypoxia in response to zoxazolamine, pentobarbitone and thiopentone sodium. Pharmacokinetics of acetyl salicylic acid, gentamicin, phenobarbitone and acetazolamide showed increase in plasma half life (t 1/2), decrease in elimination rate constant (k el) and hence prolonged residence of these drugs in hypoxic animals. INTERPRETATION & CONCLUSIONS: This experimental study showed that hypoxia altered therapeutic effectiveness and clearance of several drugs, in rats and rabbits exposed to intermittent hypobaric hypoxia. s0 uch studies need to be done in human volunteers to see the effect of hypoxia on pharmacokinetics of some common drugs.

Role of high-fat diet in regulation of gene expression of drug metabolizing enzymes and transporters.

AIM: Our aim is to investigate the molecular mechanism of regulation of gene expression of drug metabolizing enzymes (DMEs) and transporters in diet-induced obesity. MAIN METHODS: Adult male CD1 mice were fed diets containing 60% kcal fat (HFD) or 10% kcal fat (LFD) for 14 weeks. RNA levels of hepatic DMEs, transporters and their regulatory nuclear receptors (NRs) were analyzed by real-time PCR. Activation of cell-signaling components (JNK and NF-κΒ) and pro-inflammatory cytokines (IL-1ß, IL-6 and TNFα) were measured in the liver. Finally, the pharmacodynamics of drugs metabolized by DMEs was measured to determine the clinical relevance of our findings. KEY FINDINGS: RNA levels of the hepatic phase I (Cyp3a11, Cyp2b10, Cyp2a4) and phase II (Ugt1a1, Sult1a1, Sultn) enzymes were reduced ~30-60% in HFD compared to LFD mice. RNA levels of Cyp2e1, Cyp1a2 and the drug transporters, multidrug resistance proteins, (Mrp)2, Mrp3 and multidrug resistant gene (Mdr)1b were unaltered in HFD mice. Gene expression of the NRs, PXR and CAR and nuclear protein levels of RXRα was reduced in HFD mice. Cytokines, JNK and NF-κΒ were induced in HFD mice. Thus reduction in hepatic gene expression in obesity may be modulated by cross-talk between NRs and inflammation-induced cell-signaling. Sleep time of Midazolam (Cyp3a substrate) was prolonged in HFD mice, while Zoxazolamine (Cyp1a2 and Cyp2e1 substrate)-induced sleep time was unaltered. SIGNIFICANCE: This study demonstrates that gene-specific reductions in DMEs can affect specific drugs metabolized by these enzymes, thus providing a rationale to monitor the effectiveness of drug therapy in obese individuals.

Inhibitory effect of magnolol on Trp-P-2-induced DNA damage in various organs in mice.

Magnolol has been reported to strongly inhibit the mutagenicity induced by indirect mutagens in the Ames test as well as the clastogenicity induced by benzo(a)pyrene (B(a)P) in the mice micronucleus test. Here, we evaluated the inhibitory effect of magnolol on the DNA damage induced by 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in various organs using the mice alkaline single cell gel electrophoresis (SCG) assay. Animals were treated with a single oral administration of magnolol (0.01, 0.1, 1, 10, and 100 mg/kg), followed by a single intraperitoneal injection of Trp-P-2 (10 mg/kg). The liver, lung, and kidney were removed at 3 h after treatment and used in SCG assay. The results indicated that magnolol inhibited Trp-P-2-induced DNA damage in various organs. To elucidate the mechanism of this inhibitory effect against Trp-P-2, we investigated the inhibitory effect of magnolol on in vivo CYP1A2 activity using the zoxazolamine paralysis test. Magnolol significantly prolonged zoxazolamine paralysis time and showed an inhibitory effect on in vivo CYP1A2 activity. These results indicate that magnolol has an inhibitory effect on the DNA damage induced by Trp-P-2 in various organs in vivo. This inhibitory mechanism is considered due to in vivo CYP1A2 inhibition.

Sexually dimorphic regulation and induction of P450s by the constitutive androstane receptor (CAR).

The constitutive androstane receptor (CAR) is a xenosensing nuclear receptor and regulator of cytochrome P450s (CYPs). However, the role of CAR as a basal regulator of CYP expression nor its role in sexually dimorphic responses have been thoroughly studied. We investigated basal regulation and sexually dimorphic regulation and induction by the potent CAR activator TCPOBOP and the moderate CAR activator Nonylphenol (NP). NP is an environmental estrogen and one of the most commonly found environmental toxicants in Europe and the United States. Previous studies have demonstrated that NP induces several CYPs in a sexually dimorphic manner, however the role of CAR in regulating NP-mediated sexually dimorphic P450 expression and induction has not been elucidated. Therefore, wild-type and CAR-null male and female mice were treated with honey as a carrier, NP, or TCPOBOP and CYP expression monitored by QPCR and Western blotting. CAR basally regulates the expression of Cyp2c29, Cyp2b13, and potentially Cyp2b10 as demonstrated by QPCR. Furthermore, we observed a shift in the testosterone 6alpha/15alpha-hydroxylase ratio in untreated CAR-null female mice to the male pattern, which indicates an alteration in androgen status and suggests a role for androgens as CAR inverse agonists. Xenobiotic-treatments with NP and TCPOBOP induced Cyp2b10, Cyp2c29, and Cyp3a11 in a CAR-mediated fashion; however NP only induced these CYPs in females and TCPOBOP induced these CYPs in both males and females. Interestingly, Cyp2a4, was only induced in wild-type male mice by TCPOBOP suggesting Cyp2a4 induction is not sensitive to CAR-mediated induction in females. Overall, TCPOBOP and NP show similar CYP induction profiles in females, but widely different profiles in males potentially related to lower sensitivity of males to either indirect or moderate CAR activators such as NP. In summary, CAR regulates the basal and chemically inducible expression of several sexually dimorphic xenobiotic metabolizing P450s in a manner that varies depending on the ligand.

Restoration of chaperone-mediated autophagy in aging liver improves cellular maintenance and hepatic function.

Chaperone-mediated autophagy (CMA), a selective mechanism for degradation of cytosolic proteins in lysosomes, contributes to the removal of altered proteins as part of the cellular quality-control systems. We have previously found that CMA activity declines in aged organisms and have proposed that this failure in cellular clearance could contribute to the accumulation of altered proteins, the abnormal cellular homeostasis and, eventually, the functional loss characteristic of aged organisms. To determine whether these negative features of aging can be prevented by maintaining efficient autophagic activity until late in life, in this work we have corrected the CMA defect in aged rodents. We have generated a double transgenic mouse model in which the amount of the lysosomal receptor for CMA, previously shown to decrease in abundance with age, can be modulated. We have analyzed in this model the consequences of preventing the age-dependent decrease in receptor abundance in aged rodents at the cellular and organ levels. We show here that CMA activity is maintained until advanced ages if the decrease in the receptor abundance is prevented and that preservation of autophagic activity is associated with lower intracellular accumulation of damaged proteins, better ability to handle protein damage and improved organ function.

Transcription coactivator PRIP, the peroxisome proliferator-activated receptor (PPAR)-interacting protein, is redundant for the function of nuclear receptors PParalpha and CAR, the constitutive androstane receptor, in mouse liver.

Disruption of the genes encoding for the transcription coactivators, peroxisome proliferator-activated receptor (PPAR)-interacting protein (PRIP/ASC-2/RAP250/TRBP/NRC) and PPAR-binding protein (PBP/TRAP220/DRIP205/MED1), results in embryonic lethality by affecting placental and multiorgan development. Targeted deletion of coactivator PBP gene in liver parenchymal cells (PBP(LIV-/-)) results in the near abrogation of the induction of PPARalpha and CAR (constitutive androstane receptor)-regulated genes in liver. Here, we show that targeted deletion of coactivator PRIP gene in liver (PRIP(LIV-/-)) does not affect the induction of PPARalpha-regulated pleiotropic responses, including hepatomegaly, hepatic peroxisome proliferation, and induction of mRNAs of genes involved in fatty acid oxidation system, indicating that PRIP is not essential for PPARalpha-mediated transcriptional activity. We also provide additional data to show that liver-specific deletion of PRIP gene does not interfere with the induction of genes regulated by nuclear receptor CAR. Furthermore, disruption of PRIP gene in liver did not alter zoxazolamine-induced paralysis, and acetaminophen-induced hepatotoxicity. Studies with adenovirally driven EGFP-CAR expression in liver demonstrated that, unlike PBP, the absence of PRIP does not prevent phenobarbital-mediated nuclear translocation/retention of the receptor CAR in liver in vivo and cultured hepatocytes in vitro. These results show that PRIP deficiency in liver does not interfere with the function of nuclear receptors PPARalpha and CAR. The dependence of PPARalpha- and CAR-regulated gene transcription on coactivator PBP but not on PRIP attests to the existence of coactivator selectivity in nuclear receptor function.

The environmental estrogen, nonylphenol, activates the constitutive androstane receptor.

Nonylphenol (NP) and its parent compounds, the nonylphenol ethoxylates are some of the most prevalent chemicals found in U.S. waterways. NP is also resistant to biodegradation and is a known environmental estrogen, which makes NP a chemical of concern. Our data show that NP also activates the constitutive androstane receptor (CAR), an orphan nuclear receptor important in the induction of detoxification enzymes, including the P450s. Transactivation assays demonstrate that NP increases murine CAR (mCAR) transcriptional activity, and NP treatment can overcome the inhibitory effects of the inverse agonist, androstanol, on mCAR activation. Treatment of wild-type (CAR +/+) mice with NP at 50 or 75 mg/kg/day increases Cyp2b protein expression in a dose-dependent manner as demonstrated by Western blotting, and was confirmed by quantitative reverse transcription-PCR of Cyp2b10 transcript levels. CAR-null (CAR -/-) mice show no increased expression of Cyp2b following NP treatment, indicating that CAR is required for NP-mediated Cyp2b induction. In addition, NP increases the translocation of CAR into the nucleus, which is the key step in the commencement of CAR's transcriptional activity. NP also induced CYP2B6 in primary human hepatocytes, and increased Cyp2b10 messenger RNA and protein expression in humanized CAR mice, indicating that NP is an activator of human CAR as well. In conclusion, NP is a CAR activator, and this was demonstrated in vitro with transactivation assays and in vivo with transgenic CAR mouse models.

Isolation, structure elucidation and in vivo hepatoprotective potential of trans-tetracos-15-enoic acid from Indigofera tinctoria Linn.

The bioassay guided fractionation of the dried aerial part of Indigofera tinctoria Linn. led to the identification of an active fraction labelled as indigotin. On further chemical analysis, a compound isolated from indigotin was identified and characterized as trans-tetracos-15-enoic acid (TCA). The chemical structure of this compound was established on the basis of physical properties and spectral data, including NMR. It afforded significant hepatoprotection against carbon tetrachloride and paracetamol induced hepatotoxicity in experimental models. Silymarin, a well known plant based hepatoprotective agent, and N-acetylcysteine, which has proven efficacy as a replenisher of sulfhydryls, were used for relative efficacy. TCA was found to reverse the altered hepatic parameters in experimental liver damage. In the safety evaluation study the oral LD50 was found to be more than 2000 mg/kg, with no signs of abnormalities or any mortality for the 15 day period of observation after administration of a single dose of drug in mice. The studies revealed significant and concentration dependent hepatoprotective potential of TCA as it reversed the majority of the altered hepatic parameters in experimental liver damage in rats and mice and may be useful in the management of liver disorders.

Gender-specific induction of cytochrome P450s in nonylphenol-treated FVB/NJ mice.

Nonylphenol (NP) is a breakdown product of nonylphenol ethoxylates, which are used in a variety of industrial, agricultural, household cleaning, and beauty products. NP is one of the most commonly found toxicants in the United States and Europe and is considered a toxicant of concern because of its long half-life. NP is an environmental estrogen that also activates the pregnane X-receptor (PXR) and in turn induces P450s. No study to date has examined the gender-specific effects of NP on hepatic P450 expression. We provided NP at 0, 50 or 75 mg/kg/day for 7 days to male and female FVB/NJ mice and compared their P450 expression profiles. Q-PCR was performed on hepatic cDNA using primers to several CYP isoforms regulated by PXR or its relative, the constitutive androstane receptor (CAR). In female mice, NP induced Cyp2b10 and Cyp2b13, and downregulated the female-specific P450s, Cyp3a41 and Cyp3a44. In contrast, male mice treated with NP showed increased expression of Cyp2a4, Cyp2b9, and Cyp2b10. Western blots confirmed induction of Cyp2b subfamily members in both males and females. Consistent with the Q-PCR data, Western blots showed dose-dependent downregulation of Cyp3a only in females and induction of Cyp2a only in males. The overall increase in female-predominant P450s in males (Cyp2a4, 2b9) and the decrease in female-predominant P450s in females (Cyp3a41, 3a44) suggest that NP is in part feminizing the P450 profile in males and masculinizing the P450 profile in females. Testosterone hydroxylation was also altered in a gender-specific manner, as testosterone 16alpha-hydroxylase activity was only induced in NP-treated males. In contrast, NP-treated females demonstrated a greater propensity for metabolizing zoxazolamine probably due to greater Cyp2b induction in females. In conclusion, NP causes gender-specific P450 induction and therefore exposure to NP may cause distinct pharmacological and toxicological effects in males compared to females.

Transcription coactivator peroxisome proliferator-activated receptor-binding protein/mediator 1 deficiency abrogates acetaminophen hepatotoxicity.

Peroxisome proliferator-activated receptor-binding protein (PBP), also known as thyroid hormone receptor-associated protein 220/vitamin D receptor-interacting protein 205/mediator 1, an anchor for multisubunit mediator transcription complex, functions as a transcription coactivator for nuclear receptors. Disruption of the PBP gene results in embryonic lethality around embryonic day 11.5 by affecting placental and multiorgan development. Here, we report that targeted deletion of PBP in liver parenchymal cells (PBP(Liv-/-)) results in the abrogation of hypertrophic and hyperplastic influences in liver mediated by constitutive androstane receptor (CAR) ligands phenobarbital (PB) and 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene, and of acetaminophen-induced hepatotoxicity. CAR interacts with the two nuclear receptor-interacting LXXLL (L, leucine; X, any amino acid) motifs in PBP in a ligand-dependent manner. We also show that PBP interacts with the C-terminal portion of CAR, suggesting that PBP is involved in the regulation of CAR function. Although the full-length PBP only minimally increased CAR transcriptional activity, a truncated form of PBP (amino acids 487-735) functioned as a dominant negative repressor, establishing that PBP functions as a coactivator for CAR. A reduction in CAR mRNA and protein level observed in PBP(Liv-/-) mouse liver suggests that PBP may regulate hepatic CAR expression. PBP-deficient hepatocytes in liver failed to reveal PB-dependent translocation of CAR to the nucleus. Adenoviral reconstitution of PBP in PBP(Liv-/-) mouse livers restored PB-mediated nuclear translocation of CAR as well as inducibility of CYP1A2, CYP2B10, CYP3A11, and CYP7A1 expression. We conclude that transcription coactivator PBP/TRAP220/MED1 is involved in the regulation of hepatic CAR function and that PBP deficiency in liver abrogates acetaminophen hepatotoxicity.

Effect of p-amino-diphenyl ethers on hepatic microsomal cytochrome P450.

The present paper aims to investigate whether p-amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether are inhibitors as well as inducers of P450. Mice were given daily intraperitoneal (ip) injections of p-amino-2',4'-dichlorodiphenyl ether (0.25 mmol/kg) or p-amino-4'-methyldiphenyl ether (0.25 mmol/kg) for 4 days and tested at 24 h and 48 h after the last dose injection. The results showed the mice pentobarbital sleeping time was shorter and the P450 content of hepatic microsome increased significantly in the group pretreated with p-amino-4'-methyldiphenyl ether when compared with the control group, while in mice pretreated with p-amino-2',4'-dichlorodiphenyl ether the hepatic microsome P450 content increased but the pentobarbital sleeping time was extended in clear contrast to the control group. The sleeping time of the phenobarbital group (80 mg/kg daily ip injection for 4 days) was shortened at 24 h after the last injection with increased P450 content of hepatic microsome, but it showed no difference at 48 h. The zoxazolamine-paralysis times of mice treated with p-amino-2',4'-dichlorodiphenyl ether were longer than those of the control mice, while the same dose of zoxazolamine did not lead to paralysis in mice pretreated with BNF. p-Amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether inhibited the activity of 7-ethoxyresorufin O-deethylase from rat hepatic microsome induced by BNF in vitro by 70.0% and 50.1% respectively. These results suggest that p-amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether are inhibitors as well as inducers of P450.

Effect of novel anti-inflammatory ethanolamine derivatives with antioxidant properties on drug metabolising enzymes.

The influence of four ethanolamine derivatives with anti-inflammatory and antioxidant activity on the in vitro aminopyrine N-demethylation was studied. It was found that these compounds inhibit the N-demethylation of aminopyrine. 1-Cyclohexyl-5-(2-hydroxy-ethylamino)-pentan-2-one (compound 4), possessing the highest inhibitory activity and found earlier to be a potent anti-inflammatory agent, is further tested in vivo on zoxazolamine-induced paralysis, after a single administration to rats, and on aminopyrine N-demethylation, rat hepatic total cytochrome P450 and protein (postmitochondrial and microsomal) content, after a prolonged treatment. It was found that the examined compound had no significant influence on the above biotransformations, however, it could decrease the catalytically active hepatic cytochrome P450 content. These results, considered together with some structural and physicochemical properties of the compound, indicate that this compound may act as a CYP2D6 substrate.

Modulation of recombinant small-conductance Ca(2+)-activated K(+) channels by the muscle relaxant chlorzoxazone and structurally related compounds.

Using the patch clamp technique we investigated the effects of the centrally acting muscle relaxant chlorzoxazone and three structurally related compounds, 1-ethyl-2-benzimidazolinone (1-EBIO), zoxazolamine, and 1,3-dihydro-1-[2-hydroxy-5-(triflu oromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS 1619) on recombinant rat brain SK2 channels (rSK2 channels) expressed in HEK293 mammalian cells. SK channels are small conductance K(+) channels normally activated by a rise in intracellular Ca(2+) concentration; they modulate the electrical excitability in neurons and neuroendocrine cells. When applied externally, chlorzoxazone, 1-EBIO, and zoxazolamine activated rSK2 channel currents in cells dialyzed with a nominally Ca(2+)-free intracellular solution. The activation was reversible, reproducible, and depended on the chemical structure and concentration. The order of potency was 1-EBIO > chlorzoxazone > zoxazolamine. Activation of rSK2 channels by chlorzoxazone, 1-EBIO, and zoxazolamine declined at higher drug concentrations. Zoxazolamine, when applied in combination with chlorzoxazone or 1-EBIO, partially inhibited the rSK2 channel current responses, suggesting a partial-agonist mode of action. 1-EBIO failed to activate rSK2 channel currents when applied to excised inside-out membrane patches exposed to a Ca(2+)-free intracellular solution. In contrast, 1-EBIO activated rSK2 currents in a concentration-dependent manner when coapplied to the patches with a solution containing 20 nM free Ca(2+). NS 1619 did not activate rSK2 channel currents; it inhibited rSK2 channel currents activated by the other three test compounds or by high intracellular Ca(2+). We conclude that chlorzoxazone and its derivatives act through a common mechanism to modulate rSK2 channels, and SK channel modulation in the brain may partly underlie the clinical effects of chlorzoxazone.

2-Amino-5-chloro-1,3-benzoxazol-3-ium 2-(3,4-dichlorophenoxy)acetate.

The 1:1 organic salt of the title compound, C(7)H(6)ClN(2)O(+). C(8)H(5)Cl(2)O(3)(-) or [(2-ABOX)(3,4-D)], comprises the two constituent molecules associated by an R(2)(2)(8) graph-set interaction through the carboxylate group of 3,4-D across the protonated N/N sites of 2-ABOX [N.O 2.546 (3) and 2.795 (3) A]. Cation/anion pairs associate across an inversion centre forming discrete tetramers via an additional three-centre hydrogen-bonding association from the latter N amino proton to a phenoxy O atom [N.O 3.176 (3) A] and a carboxylate O atom [N.O 2.841 (3) A]. This formation differs from the polymeric hydrogen-bonded chains previously observed for adduct structures of 2-ABOX with carboxylic acids.

The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism.

Organisms encounter a wide range of foreign compounds--or 'xenobiotics'--with potentially harmful consequences. The cytochrome P450 (CYP) enzymes metabolize xenobiotics and thus are a primary defence against these compounds. Increased expression of specific CYP genes in response to particular xenobiotics is a central component of this defence, although such induction can also increase production of toxic metabolites. Here we show that the nuclear receptor CAR mediates the response evoked by a class of xenobiotics known as the 'phenobarbital-like inducers'. The strong activation of Cyp2b10 gene expression by phenobarbital, or by the more potent TCPOBOP, is absent in mice lacking the CAR gene. These animals also show decreased metabolism of the classic CYP substrate zoxazolamine and a complete loss of the liver hypertrophic and hyperplastic responses to these inducers. Cocaine causes acute hepatotoxicity in wild-type mice previously exposed to phenobarbital-like inducers and this toxicity is also absent in the CAR-deficient animals. Thus, loss of CAR function alters sensitivity to toxins, increasing or decreasing it depending on the compound. Modulation of CAR activity in humans may significantly affect metabolism of drugs and other xenobiotics.

Pharmacological activation of cloned intermediate- and small-conductance Ca(2+)-activated K(+) channels.

We previously characterized 1-ethyl-2-benzimidazolinone (1-EBIO), as well as the clinically useful benzoxazoles, chlorzoxazone (CZ), and zoxazolamine (ZOX), as pharmacological activators of the intermediate-conductance Ca(2+)-activated K(+) channel, hIK1. The mechanism of activation of hIK1, as well as the highly homologous small-conductance, Ca(2+)-dependent K(+) channel, rSK2, was determined following heterologous expression in Xenopus oocytes using two-electrode voltage clamp (TEVC) and excised, inside-out patch-clamp techniques. 1-EBIO, CZ, and ZOX activated both hIK1 and rSK2 in TEVC and excised inside-out patch-clamp experiments. In excised, inside-out patches, 1-EBIO and CZ induced a concentration-dependent activation of hIK1, with half-maximal (K(1/2)) values of 84 microM and 98 microM, respectively. Similarly, CZ activated rSK2 with a K(1/2) of 87 microM. In the absence of CZ, the Ca(2+)-dependent activation of hIK1 was best fit with a K(1/2) of 700 nM and a Hill coefficient (n) of 2.0. rSK2 was activated by Ca(2+) with a K(1/2) of 700 nM and an n of 2.5. Addition of CZ had no effect on either the K(1/2) or n for Ca(2+)-dependent activation of either hIK1 or rSK2. Rather, CZ increased channel activity at all Ca(2+) concentrations (V(max)). Event-duration analysis revealed hIK1 was minimally described by two open and three closed times. Activation by 1-EBIO had no effect on tau(o1), tau(o2), or tau(c1), whereas tau(c2) and tau(c3) were reduced from 9.0 and 92.6 ms to 5.0 and 44.1 ms, respectively. In conclusion, we define 1-EBIO, CZ, and ZOX as the first known activators of hIK1 and rSK2. Openers of IK and SK channels may be therapeutically beneficial in cystic fibrosis and vascular diseases.

Stimulation of Cl(-) secretion by chlorzoxazone.

We previously demonstrated that 1-ethyl-2-benzimidazolone (1-EBIO) directly activates basolateral membrane calcium-activated K(+) channels (K(Ca)), thereby stimulating Cl(-) secretion across several epithelia. In our pursuit to identify potent modulators of Cl(-) secretion that may be useful to overcome the Cl(-) secretory defect in cystic fibrosis (CF), we have identified chlorzoxazone [5-chloro-2(3H)-benzoxazolone], a clinically used centrally acting muscle relaxant, as a stimulator of Cl(-) secretion in several epithelial cell types, including T84, Calu-3, and human bronchial epithelium. The Cl(-) secretory response induced by chlorzoxazone was blocked by charybdotoxin (CTX), a known blocker of K(Ca). In nystatin-permeabilized monolayers, chlorzoxazone stimulated a basolateral membrane I(K), which was inhibited by CTX and also stimulated an apical I(Cl) that was inhibited by glibenclamide, indicating that the G(Cl) responsible for this I(Cl) may be cystic fibrosis transmembrane conductance regulator (CFTR). In membrane vesicles prepared from T84 cells, chlorzoxazone stimulated (86)Rb(+) uptake in a CTX-sensitive manner. In excised, inside-out patches, chlorzoxazone activated an inwardly-rectifying K(+) channel, which was inhibited by CTX. 6-Hydroxychlorzoxazone, the major metabolite of chlorzoxazone, did not activate K(Ca), whereas zoxazolamine (2-amino-5-chlorzoxazole) showed a similar response profile as chlorzoxazone. In normal human nasal epithelium, chlorzoxazone elicited hyperpolarization of the potential difference that was similar in magnitude to isoproterenol. However, in the nasal epithelium of CF patients with the DeltaF508 mutation of CFTR, there was no detectable Cl(-) secretory response to chlorzoxazone. These studies demonstrate that chlorzoxazone stimulates transepithelial Cl(-) secretion in normal airway epithelium in vitro and in vivo, and suggest that stimulation requires functional CFTR in the epithelia.

Acute and subchronic evaluation of Indigofera arrecta: absence of both toxicity and modulation of selected cytochrome P450 isozymes in ddY mice.

Indigofera arrecta, an anti diabetic plant was investigated in ddY mice to determine its acute and subchronic effects, and whether it modulated hepatic cytochrome P450 (CYP) isozymes and glutathione (GSH). No mortality was observed in the acute (up to 10 g I. arrecta/kg body wt, p.o.) and subchronic (2 g I. arrecta/kg body wt, p.o. daily for 30 days) studies. The extract did not alter haematological indices, serum and tissue lipids and glutathione but lowered serum bile acids. The latter phenomenon is under further investigation. Neither the duration of pentobarbital (PB) and zoxazolamine (ZA) effects in vivo, nor CYP-dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-depentylase (PROD) and p-nitrophenol hydroxylase (PNPH) activities in vitro were altered by I. arrecta. The extract was thus devoid of overt acute and subchronic toxic effects, and did not affect CYPs and GSH whose modulation may cause interactions of components in a multiple drug therapy.