Molecular Pharmacology most recent issues

[ARTICLES] Comparison of the Potential Multiple Binding Modes of Bicyclam, Monocylam, and Noncyclam Small-Molecule CXC Chemokine Receptor 4 Inhibitors

Wong, R. S. Y., Bodart, V., Metz, M., Labrecque, J., Bridger, G., Fricker, S. P. — 2008-11-17

CXC chemokine receptor (CXCR)4 is an HIV coreceptor and a chemokine receptor that plays an important role in several physiological and pathological processes, including hematopoiesis, leukocyte homing and trafficking, metastasis, and angiogenesis. This receptor belongs to the class A family of G protein-coupled receptors and is a validated target for the development of a new class of antiretroviral therapeutics. This study compares the interactions of three structurally diverse small-molecule CXCR4 inhibitors with the receptor and is the first report of the molecular interactions of the nonmacrocyclic CXCR4 inhibitor (S)-N'-(1H-benzimidazol-2-ylmethyl)-N'-(5,6,7,8-tetrahydroquinolin-8-yl)butene-1,4-diamine (AMD11070). Fourteen CXCR4 single-site mutants representing amino acid residues that span the entire putative ligand binding pocket were used in this study. These mutants were used in binding studies to examine how each single-site mutation affected the ability of the inhibitors to compete with ¹²⁵I-stromal-derived factor-1 binding. Our data suggest that these CXCR4 inhibitors bind to overlapping but not identical amino acid residues in the transmembrane regions of the receptor. In addition, our results identified amino acid residues that are involved in unique interactions with two of the CXCR4 inhibitors studied. These data suggest an extended binding pocket in the transmembrane regions close to the second extracellular loop of the receptor. Based on site-directed mutagenesis and molecular modeling, several potential binding modes were proposed for each inhibitor. These mechanistic studies might prove to be useful for the development of future generations of CXCR4 inhibitors with improved clinical pharmacology and safety profiles.

[ARTICLES] Multiple Pharmacophores for the Selective Activation of Nicotinic {alpha}7-Type Acetylcholine Receptors

Horenstein, N. A., Leonik, F. M., Papke, R. L. — 2008-11-17

The activation of heteromeric and homomeric nicotinic acetylcholine receptors was studied in Xenopus laevis oocytes to identify key structures of putative agonist molecules associated with the selective activation of homomeric 7 receptors. We observed that selectivity between 7 and 4β2 was more readily obtained than selectivity between 7 and 3β4. Based on structural comparisons of previously characterized selective and nonselective agonists, we hypothesize at least three chemical motifs exist that, when present in molecules containing an appropriate cationic center, could be associated with the selective activation of 7 receptors. We identify the three distinct structural motifs based on prototypical drugs as the choline motif, the tropane motif, and the benzylidene motif. The choline motif involves the location of an oxygen-containing polar group such as a hydroxyl or carbonyl separated by two carbons from the charged nitrogen. The tropane motif provides 7-selectivity based on the addition of multiple small hydrophobic groups positioned away from the cationic center in specific orientations. We show that this motif can convert the nonselective agonists quinuclidine and ethyltrimethyl-ammonium to the 7-selective analogs methyl-quinuclidine and diethyldimethyl-ammonium, respectively. We have shown previously that the benzylidene group of 3-2,4, dimethoxy-benzylidene anabaseine (GTS-21) converts anabaseine into an 7-selective agonist. The benzylidene motif was also applied to quinuclidine to generate another distinct family of 7-selective agonists. Our results provide insight for the further development of nicotinic therapeutics and will be useful to direct future experiments with protein structure-based modeling and site-directed mutagenesis.

[ARTICLES] Structural Basis of Human Pregnane X Receptor Activation by the Hops Constituent Colupulone

Teotico, D. G., Bischof, J. J., Peng, L., Kliewer, S. A., Redinbo, M. R. — 2008-11-17

Hops extracts are used to alleviate menopausal symptoms and as an alternative to hormone replacement therapy, but they can produce potentially harmful drug-drug interactions. The nuclear xenobiotic receptor pregnane X receptor (PXR) is promiscuously activated by a range of structurally distinct chemicals. It has a key role in the transcriptional regulation of genes that encode xenobiotic metabolism enzymes. In this study, hops extracts are shown to induce the expression of numerous drug metabolism and excretion proteins. The β-bitter acid colupulone is demonstrated to be a bioactive component and direct activator of human PXR. The 2.8-Å resolution crystal structure of the ligand binding domain of human PXR in complex with colupulone was elucidated, and colupulone was observed to bind in a single orientation stabilized by both van der Waals and hydrogen bonding contacts. The crystal structure also indicates that related - and β-bitter acids have the capacity to serve as PXR agonists as well. Taken together, these results reveal the structural basis for drug-drug interactions mediated by colupulone and related constituents of hops extracts.

[ARTICLES] The Orphan Transporter Rxt1/NTT4 (SLC6A17) Functions as a Synaptic Vesicle Amino Acid Transporter Selective for Proline, Glycine, Leucine, and Alanine

2008-11-17

Rxt1/NTT4 (SLC6A17) belongs to a gene family of "orphan transporters" whose substrates and consequently functions remain unidentified. Although Rxt1/NTT4 was previously thought to function as a sodium-dependent plasma membrane transporter, recent studies localized the protein to synaptic vesicles of glutamatergic and GABAergic neurons. Here, we provide evidence indicating that Rxt1/NTT4 functions as a vesicular transporter selective for proline, glycine, leucine, and alanine. Using Western blot, immunoprecipitation, immunocytochemistry, and polymerase chain reaction approaches, we demonstrate that PC12 cells express the Rxt1/NTT4 gene and protein. Small interfering RNA (siRNA)-mediated knockdown of Rxt1/NTT4 in PC12 cells resulted in selective reductions in uptake levels for proline, glycine, leucine, and alanine. Likewise, gas chromatography analysis of amino acid content in an enriched synaptic vesicle fraction from wild-type and siRNA-Rxt1/NTT4 PC12 cells revealed that proline, glycine, leucine, and alanine levels were decreased in siRNA-treated cells compared with wild-type cells. Furthermore, Rxt1/NTT4-transfected Chinese hamster ovary (CHO) cells exhibited significant uptake increases of these amino acids compared with mock-transfected CHO cells. Finally, proline uptake in both PC12 cells and Rxt1/NTT4-transfected CHO cells was dependent on the electrochemical gradient maintained by the vacuolar-type H⁺-ATPase. These data indicate that the orphan Rxt1/NTT4 protein functions as a vesicular transporter for proline, glycine, leucine, and alanine, further suggesting its important role in synaptic transmission.

[ARTICLES] 17-{beta}-Estradiol Inhibits Transforming Growth Factor-{beta} Signaling and Function in Breast Cancer Cells via Activation of Extracellular Signal-Regulated Kinase through the G Protein-Coupled Receptor 30

Kleuser, B., Malek, D., Gust, R., Pertz, H. H., Potteck, H. — 2008-11-17

Breast cancer development and breast cancer progression involves the deregulation of growth factors leading to uncontrolled cellular proliferation, invasion and metastasis. Transforming growth factor (TGF)-β plays a crucial role in breast cancer because it has the potential to act as either a tumor suppressor or a pro-oncogenic chemokine. A cross-communication between the TGF-β signaling network and estrogens has been postulated, which is important for breast tumorigenesis. Here, we provide evidence that inhibition of TGF-β signaling is associated with a rapid estrogen-dependent nongenomic action. Moreover, we were able to demonstrate that estrogens disrupt the TGF-β signaling network as well as TGF-β functions in breast cancer cells via the G protein-coupled receptor 30 (GPR30). Silencing of GPR30 in MCF-7 cells completely reduced the ability of 17-β-estradiol (E2) to inhibit the TGF-β pathway. Likewise, in GPR30-deficient MDA-MB-231 breast cancer cells, E2 achieved the ability to suppress TGF-β signaling only after transfection with GPR30-encoding plasmids. It is most interesting that the antiestrogen fulvestrant (ICI 182,780), which possesses agonistic activity at the GPR30, also diminished TGF-β signaling. Further experiments attempted to characterize the molecular mechanism by which activated GPR30 inhibits the TGF-β pathway. Our results indicate that GPR30 induces the stimulation of the mitogen-activated protein kinases (MAPKs), which interferes with the activation of Smad proteins. Inhibition of MAPK activity prevented the ability of E2 from suppressing TGF-β signaling. These findings are of great clinical relevance, because down-regulation of TGF-β signaling is associated with the development of breast cancer resistance in response to antiestrogens.

[ARTICLES] Aryl Hydrocarbon Receptor-Mediated Down-Regulation of Sox9b Causes Jaw Malformation in Zebrafish Embryos

Xiong, K. M., Peterson, R. E., Heideman, W. — 2008-11-17

Exposure to environmental contaminants can disrupt normal development of the early vertebrate skeleton. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) impairs craniofacial skeletal development across many vertebrate species, and its effects are especially prominent in early life stages of fish. TCDD activates the aryl hydrocarbon receptor, a transcription factor that mediates most if not all TCDD responses. We investigated the transcriptional response in the developing zebrafish jaw after TCDD exposure using DNA microarrays. Zebrafish larvae were exposed to TCDD at 96 h after fertilization, and jaw cartilage tissue was harvested for microarray analysis at 1, 2, 4, and 12 h after exposure. Numerous chondrogenic transcripts were misregulated by TCDD in the jaw. Comparison of transcripts altered by TCDD in jaw with transcripts altered in embryonic heart showed that the transcriptional responses in the jaw and the heart were strikingly different. Sox9b, a critical chondrogenic transcription factor, was the most significantly reduced transcript in the jaw. We hypothesized that the TCDD reduction of sox9b expression plays an integral role in affecting the formation of the embryonic jaw. Morpholino knockdown of sox9b expression demonstrated that partial reduction of sox9b expression alone was sufficient to produce a TCDD-like jaw phenotype. Loss of a single copy of the sox9b gene in sox9b(+/-) heterozygotes increased sensitivity to jaw malformation by TCDD. Finally, embryos injected with sox9b mRNA and then exposed to TCDD blocked TCDD-induced jaw toxicity in approximately 14% of sox9b-injected embryos. These results suggest that reduced sox9b expression in TCDD-exposed zebrafish embryos contributes to jaw malformation.

[ARTICLES] Different Interactions between MT7 Toxin and the Human Muscarinic M1 Receptor in Its Free and N-Methylscopolamine-Occupied States

2008-11-17

Muscarinic MT7 toxin is a highly selective and potent antagonist of the M₁ subtype of muscarinic receptor and acts by binding to an allosteric site. To identify the molecular determinants by which MT7 toxin interacts with this receptor in its free and NMS-occupied states, the effect on toxin potency of alanine substitution was evaluated in equilibrium and kinetic binding experiments as well as in functional assays. The determination of the crystallographic structure of an MT7-derivative (MT7-diiodoTyr51) allowed the selection of candidate residues that are accessible and present on both faces of the three toxin loops. The equilibrium binding data are consistent with negative cooperativity between N-methylscopolamine (NMS) and wild-type or modified MT7 and highlight the critical role of the tip of the central loop of the toxin (Arg34, Met35 Tyr36) in its interaction with the unoccupied receptor. Examination of the potency of wild-type and modified toxins to allosterically decrease the dissociation rate of [³H]NMS allowed the identification of the MT7 residues involved in its interaction with the NMS-occupied receptor. In contrast to the results with the unoccupied receptor, the most important residue for this interaction was Tyr36 in loop II, assisted by Trp10 in loop I and Arg52 in loop III. The critical role of the tips of the MT7 loops was also confirmed in functional experiments. The high specificity of the MT7-M₁ receptor interaction exploits several MT7-specific residues and reveals a different mode of interaction of the toxin with the free and NMS-occupied states of the receptor.

[ARTICLES] Overexpression of Heme Oxygenase-1 Protects Dopaminergic Neurons against 1-Methyl-4-Phenylpyridinium-Induced Neurotoxicity

Hung, S.-Y., Liou, H.-C., Kang, K.-H., Wu, R.-M., Wen, C.-C., Fu, W.-M. — 2008-11-17

Heme oxygenase-1 (HO-1) is up-regulated in response to oxidative stress and catalyzes the degradation of pro-oxidant heme to carbon monoxide (CO), iron, and bilirubin. Intense HO-1 immunostaining in the Parkinsonian brain is demonstrated, indicating that HO-1 may be involved in the pathogenesis of Parkinsonism. We here locally injected adenovirus containing human HO-1 gene (Ad-HO-1) into rat substantia nigra concomitantly with 1-methyl-4-phenylpyridinium (MPP⁺). Seven days after injection of MPP⁺ and Ad-HO-1, the brain was isolated for immunostaining and for measurement of dopamine content and inflammatory cytokines. It was found that overexpression of HO-1 significantly increased the survival rate of dopaminergic neurons; reduced the production of tumor necrosis factor (TNF-) and interleukin-1β (IL-1β) in substantia nigra; antagonized the reduction of striatal dopamine content induced by MPP⁺; and also up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) expression in substantia nigra. Apomorphine-induced rotation after MPP⁺ treatment was also inhibited by Ad-HO-1. On the other hand, inhibition of HO enzymatic activity by zinc protoporphyrin-IX facilitated the MPP⁺-induced rotatory behavior and enhanced the reduction of dopamine content. HO-1 overexpression also protected dopaminergic neurons against MPP⁺-induced neurotoxicity in midbrain neuron-glia cocultures. Overexpression of HO-1 increased the expression of BDNF and GDNF in astrocytes and BDNF in neurons. Our results indicate that HO-1 induction exerts neuroprotection both in vitro and in vivo. Pharmacological or genetic approaches targeting HO-1 may represent a promising and novel therapeutic strategy in treating Parkinsonism.

[ARTICLES] Identification of Quinolines that Inhibit Melanogenesis by Altering Tyrosinase Family Trafficking

Ni-Komatsu, L., Tong, C., Chen, G., Brindzei, N., Orlow, S. J. — 2008-11-17

A series of quinolines, including chloroquine and quinine, were identified as potent pigmentation inhibitors through screening a compound library in murine melanocytes. Structure-activity relationship analysis indicated that 4-substituted amino groups with a tertiary amine side chain, such as chloroquine, were associated with robust inhibitory activity. In contrast to many previously identified pigmentation inhibitors, these newly identified inhibitors had no effect on either the level or the enzymatic activity of tyrosinase, the rate-limiting enzyme in melanin production. Rather, our results showed that these quinolines inhibited melanogenesis by disrupting the intracellular trafficking of tyrosinase-related proteins and lysosome-associated membrane protein 1 (Lamp-1). In treated melanocytes, tyrosinase and tyrosinase-related protein 1 accumulated in Lamp-1-positive perinuclear organelles instead of melanosomes, thus preventing melanogenesis. The depigmenting abilities of chloroquine and quinine salicylate were assessed in a human skin equivalent model (MelanoDerm). Both compounds were considerably more effective than arbutin, a widely used lightening agent. Our results indicate that quinolines may be useful agents for "cosmeceutical" skin lightening and treatment of hyperpigmentation disorders.

[ARTICLES] Imidazoquinolinone, Imidazopyridine, and Isoquinolindione Derivatives as Novel and Potent Inhibitors of the Poly(ADP-ribose) Polymerase (PARP): A Comparison with Standard PARP Inhibitors

2008-11-17

We have identified three novel structures for inhibitors of the poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA and implicated in DNA repair, apoptosis, organ dysfunction or necrosis. 2-[4-(5-Methyl-1H-imidazol-4-yl)-piperidin-1-yl]-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK49187), 2-(4-pyridin-2-yl-phenyl)-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK236864), 6-chloro-8-hydroxy-2,3-dimethyl-imidazo-[1,2-]-pyridine (BYK20370), and 4-(1-methyl-1H-pyrrol-2-ylmethylene)-4H-isoquinolin-1,3-dione (BYK204165) inhibited cell-free recombinant human PARP-1 with pIC₅₀ values of 8.36, 7.81, 6.40, and 7.35 (pK_i 7.97, 7.43, 5.90, and 7.05), and murine PARP-2 with pIC₅₀ values of 7.50, 7.55, 5.71, and 5.38, respectively. BYK49187, BYK236864, and BYK20370 displayed no selectivity for PARP-1/2, whereas BYK204165 displayed 100-fold selectivity for PARP-1. The IC₅₀ values for inhibition of poly(ADP-ribose) synthesis in human lung epithelial A549 and cervical carcinoma C4I cells as well in rat cardiac myoblast H9c2 cells after PARP activation by H₂O₂ were highly significantly correlated with those at cell-free PARP-1 (r² = 0.89-0.96, P < 0.001) but less with those at PARP-2 (r² = 0.78-0.84, P < 0.01). The infarct size caused by coronary artery occlusion and reperfusion in the anesthetized rat was reduced by 22% (P < 0.05) by treatment with BYK49187 (3 mg/kg i.v. bolus and 3 mg/kg/h i.v. during 2-h reperfusion), whereas the weaker PARP inhibitors, BYK236864 and BYK20370, were not cardioprotective. In conclusion, the imidazoquinolinone BYK49187 is a potent inhibitor of human PARP-1 activity in cell-free and cellular assays in vitro and reduces myocardial infarct size in vivo. The isoquinolindione BYK204165 was found to be 100-fold more selective for PARP-1. Thus, both compounds might be novel and valuable tools for investigating PARP-1-mediated effects.

[ARTICLES] Identification and Functional Characterization of Allosteric Agonists for the G Protein-Coupled Receptor FFA2

2008-11-17

FFA2 (GPR43) has been identified as a receptor for short-chain fatty acids (SCFAs) that include acetate and propionate. FFA2 is highly expressed in islets, a subset of immune cells, and adipocytes. Although the potential roles of FFA2 activation in these tissues have previously been described, the physiological functions are still unclear. The potency for SCFAs on FFA2 is low, in the high micromolar to millimolar concentrations. To identify better pharmacological tools to study receptor function, we used high-throughput screening (HTS) to discover a series of small molecule phenylacetamides as novel and more potent FFA2 agonists. This series is specific for FFA2 over FFA1 (GPR40) and FFA3 (GPR41), and it is able to activate both the G_q and G_i pathways in vitro on Chinese hamster ovary cells stably expressing FFA2. Treatment of adipocytes with these compounds also resulted in G_i-dependent inhibition of lipolysis similar to that of endogenous ligands (SCFAs). It is noteworthy that these compounds not only acted as FFA2 agonists but also exhibited positive cooperativity with acetate or propionate. The observed allosteric modulation was consistent in all the functional assays that we have explored, including cAMP, calcium mobilization, guanosine 5'-[-thio]triphosphate binding, and lipolysis. Molecular modeling analysis of FFA2 based on human β₂-adrenergic receptor structure revealed potential nonoverlapping binding sites for the endogenous and synthetic ligands, further providing insight into the binding pocket for the allosteric interactions. This is the first report describing the identification of novel allosteric modulators with agonist activity for FFA2, and these compounds may serve as tools for further unraveling the physiological functions of the receptor and its involvement in various diseases.

[ARTICLES] Endoplasmic Reticulum Stress Induces Leptin Resistance

2008-11-17

Leptin is an important circulating signal for inhibiting food intake and body weight gain. In recent years, "leptin resistance" has been considered to be one of the main causes of obesity. However, the detailed mechanisms of leptin resistance are poorly understood. Increasing evidence has suggested that stress signals, which impair endoplasmic reticulum (ER) function, lead to an accumulation of unfolded proteins, which results in ER stress. In the present study, we hypothesized that ER stress is involved in leptin resistance. Tunicamycin, thapsigargin, or brefeldin A was used to induce ER stress. The activation status of leptin signals was measured by Western blotting analysis using a phospho-(Tyr705) signal transducer and activator of transcription 3 (STAT3) antibody. We observed that ER stress markedly inhibited leptin-induced STAT3 phosphorylation. In contrast, ER stress did not affect leptin-induced c-Jun NH₂-terminal kinase activation. These results suggest that ER stress induces leptin resistance. ER stress-induced leptin resistance was mediated through protein tyrosine phosphatase 1B but not through suppressors of cytokine signaling 3. It is noteworthy that a chemical chaperone, which could improve the protein-folding capacity, reversed ER stress-induced leptin resistance. Moreover, homocysteine, which induces ER stress, caused leptin resistance both in vitro and in vivo. Together, these findings suggest that the pathological mechanism of leptin resistance is derived from ER stress.

[ARTICLES] Rebeccamycin Derivatives as Dual DNA-Damaging Agents and Potent Checkpoint Kinase 1 Inhibitors

2008-11-17

Rebeccamycin is an indolocarbazole class inhibitor of topoisomerase I. In the course of structure-activity relationship studies on rebeccamycin derivatives, we have synthesized analogs with the sugar moiety attached to either one or both indole nitrogens. Some analogs, especially those with substitutions at the 6' position of the carbohydrate moiety, exhibit potent inhibitory activity toward checkpoint kinase 1 (Chk1), a kinase that has a major role in the G₂/M checkpoint in response to DNA damage. Some of these compounds retained a genotoxic activity either through intercalation into the DNA and/or by topoisomerase I-mediated DNA cleavage. We explored the structure-activity relationship between these compounds and their multiple targets. These rebeccamycin derivatives represent a novel class of potential antitumor agents that have a dual effect and might selectively induce the death of cancer cells.

[ARTICLES] Mechanistic Differences between GSH Transport by Multidrug Resistance Protein 1 (MRP1/ABCC1) and GSH Modulation of MRP1-Mediated Transport

Rothnie, A., Conseil, G., Lau, A. Y. T., Deeley, R. G., Cole, S. P. C. — 2008-11-17

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent polytopic membrane protein that transports many anticancer drugs and organic anions. Its transport mechanism is multifaceted, especially with respect to the participation of GSH. For example, vincristine is cotransported with GSH, estrone sulfate transport is stimulated by GSH, or MRP1 can transport GSH alone, and this can be stimulated by compounds such as verapamil or apigenin. Thus, the interactions between GSH and MRP1 are mechanistically complex. To examine the similarities and differences among the various GSH-associated mechanisms of MRP1 transport, we have measured first the effect of GSH and several GSH-associated substrates/modulators on the binding and hydrolysis of ATP by MRP1 using 8-azidoadenosine-5'-[³²P]-triphosphate ([³²P]azidoATP) analogs, and second the initial binding of GSH and GSH-associated substrates/modulators to MRP1. We observed that GSH or its nonreducing derivative S-methylGSH (S-mGSH), but none of the GSH-associated substrate/modulators, caused a significant increase in [-³²P]azidoATP labeling of MRP1. Moreover, GSH and S-mGSH decreased levels of orthovanadate-induced trapping of [-³²P]azidoADP. [-³²P]azidoADP.Vi trapping was also decreased by estone sulfate, whereas vincristine, verapamil, and apigenin had no apparent effects on nucleotide interactions with MRP1. Furthermore, estrone sulfate and S-mGSH enhanced the effect of each other 15- and 10-fold, respectively. Second, although GSH binding increased the apparent affinity of MRP1 for all GSH-associated substrates/modulators tested, only estrone sulfate had a reciprocal effect on the apparent affinity of MRP1 for GSH. Overall, these results indicate significant mechanistic differences between MRP1-mediated transport of GSH and the ability of GSH to modulate MRP1 transport.

[ARTICLES] Nicotine Relieves Anxiogenic-Like Behavior in Mice that Overexpress the Read-Through Variant of Acetylcholinesterase but Not in Wild-Type Mice

2008-11-17

Stress increases vulnerability and causes relapse to drugs of abuse. The usually rare read-through variant of acetylcholinesterase (AChE-R) is causally involved in stress-related behaviors, and transgenic mice constitutively overexpressing AChE-R (TgR) show behaviors characteristic of chronic stress. We measured anxiety-like behavior on TgR and control mice under normal conditions and under long-term nicotine treatment. In addition, we measured epibatidine binding in the brain and transcription status in the striatum, using microarrays, in wild-type and TgR mice. TgR mice behaved as more anxious than controls, an effect normalized by long-term nicotine intake. In control mice, long-term nicotine augmented epibatidine binding in several areas of the brain, including the hippocampus and striatum. In TgR transgenics, long-term nicotine increased epibatidine binding in some areas but not in the hippocampus or the striatum. Because the striatum is involved in the mechanisms of drug addiction, we studied how the transgene affected striatal gene expression. Whole-genome DNA microarray showed that 23 transcripts were differentially expressed in TgR mouse striata, including 15 known genes, 7 of which are anxiety-related. Subsequent reverse-transcriptase polymerase chain reaction validated changes in 7 of those 15 genes, confirmed the increase trend in 5 more transcripts, and further revealed changes in 5 genes involved in cholinergic signaling. In summary, we found that nicotine acts as an anxiolytic in TgR mice but not in control mice and that continuously overexpressed AChE-R regulates striatal gene expression, modulating cholinergic signaling and stress-related pathways.

[ARTICLES] 12(R)-Hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic Acid [12(R)-HETE], an Arachidonic Acid Derivative, Is an Activator of the Aryl Hydrocarbon Receptor

Chiaro, Christopher. R., Patel, R. D., Perdew, Gary. H. — 2008-11-17

The aryl hydrocarbon receptor (AHR) is a ligand-regulated transcription factor that can be activated by structurally diverse chemicals, ranging from environmental carcinogens to dietary metabolites. Evidence supporting a necessary role for the AHR in normal biology has been established; however, identification of key endogenous ligand/activator remains to be established. Here, we report the ability of 12(R)-hydroxy-5(Z),8(Z),10(E), 14(Z)-eicosatetraenoic acid [12(R)-HETE], an arachidonic acid metabolite produced by either a lipoxygenase or cytochrome P-450 pathway, to act as a potent indirect modulator of the AHR pathway. In contrast, structurally similar HETE isomers failed to demonstrate significant activation of the AHR. Electrophoretic mobility shift assays, together with ligand competition binding experiments, have demonstrated the inability of 12(R)-HETE to directly bind or directly activate the AHR to a DNA binding species in vitro. However, cell-based xenobiotic-responsive element-driven luciferase reporter assays indicate the ability of 12(R)-HETE to modulate AHR activity, and quantitation of induction of an AHR target gene confirmed 12(R)-HETE's ability to activate AHR-mediated transcription, even at high nanomolar concentrations in human hepatoma (HepG2)- and keratinocyte (HaCaT)-derived cell lines. One explanation for these results is that a metabolite of 12(R)-HETE is acting as a direct ligand for the AHR. However, several known metabolites failed to exhibit AHR activity. The ability of 12(R)-HETE to activate AHR target genes required receptor expression. These results indicate that 12(R)-HETE can serve as a potent activator of AHR activity and suggest that in normal and inflammatory disease conditions in skin, 12(R)-HETE is produced, perhaps leading to AHR activation.

[ARTICLES] Dissecting the Role of Multiple Reductases in Bioactivation and Cytotoxicity of the Antitumor Agent 2,5-Diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1)

Yan, C., Kepa, J. K., Siegel, D., Stratford, I. J., Ross, D. — 2008-11-17

2,5-Diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1) is a novel antitumor diaziridinyl benzoquinone derivative designed to be bioactivated by the two-electron reductase NAD(P)H:quinone oxidoreductase (NQO1) and is currently in clinical trials. NQO1 is expressed at high levels in many solid tumors. RH1 cytotoxicity has been shown previously to be NQO1-dependent. The purpose of this study was to investigate whether other reducing enzymes such as cytochrome b₅ reductase (b5R), cytochrome P450 reductase (P450R), dihydronicotinamide riboside:quinone oxidoreductase 2 (NQO2), and xanthine oxidase/xanthine dehydrogenase (XO/XDH) also contribute to the bioactivation and cytotoxicity of RH1 in human tumor cells. For these studies, we established a series of stable MDA468 breast cancer cell lines overexpressing various levels of NQO1, b5R, P450R, and NQO2 and compared RH1-induced growth inhibition [3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium and sulforhodamine B analysis] and interstrand DNA cross-linking (comet analysis) in both parental MDA468 cells and transfected clones. RH1 toxicity correlated with NQO1 and NQO2 but not with either b5R or P450R activity levels in the respective series of transfected MDA468 cell clones. Enzymatic assays showed that RH1 was an in vitro substrate for xanthine oxidase. However, XO/XDH protein and activity could not be detected in a variety of human tumor cell lines. These studies suggest that NQO1 and NQO2 are the principal enzymatic determinants of RH1 bioactivation in MDA468 tumor cells and that b5R, P450R, and XDH/XO are unlikely to play major roles. Our studies also suggest that NQO2 may be particularly relevant as a bioactivation system for RH1 in NQO1-deficient tumors such as leukemias and lymphomas.

[ARTICLES] P2Y1 Receptor Activation Elicits Its Partition out of Membrane Rafts and Its Rapid Internalization from Human Blood Vessels: Implications for Receptor Signaling

2008-11-17

The nucleotide P2Y₁ receptor (P2Y₁R) is expressed in both the endothelial and vascular smooth muscle cells; however, its plasma membrane microregionalization and internalization in human tissues remain unknown. We report on the role of membrane rafts in P2Y₁R signaling by using sodium carbonate or OptiPrep sucrose density gradients, Western blot analysis, reduction of tissue cholesterol content, and vasomotor assays of endothelium-denuded human chorionic arteries. In tissue extracts prepared either in sodium carbonate or OptiPrep, approximately 20 to 30% of the total P2Y₁R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl β-cyclodextrin reduced the raft partitioning of the P2Y₁R and obliterated the P2Y₁R-mediated contractions but not the vasomotor responses elicited by either serotonin or KCl. Perfusion of chorionic artery segments with 100 nM 2-methylthio ADP or 10 nM [[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl] 2,3dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS 2365), a selective P2Y₁R agonist, not only displaced within 4 min the P2Y₁R localization out of membrane rafts but also induced its subsequent internalization. 2'-Deoxy-N⁶-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y₁R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y₁R from the membrane raft, further evidencing the pharmacodynamics of the receptor-ligand interaction. Vascular reactivity assays showed fading of the ligand-induced vasoconstrictions, a finding that correlated with the P2Y₁R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y₁R to membrane rafts, highlighting the role of this microdomain in P2Y₁R signaling.

[ARTICLES] The Cannabinoid CB1 Receptor Antagonist Rimonabant Stimulates 2-Deoxyglucose Uptake in Skeletal Muscle Cells by Regulating the Expression of Phosphatidylinositol-3-kinase

2008-11-17

The endocannabinoid system regulates food intake, energy, and glucose metabolism at both central and peripheral levels. We have investigated the mechanism by which it may control glucose uptake in skeletal muscle cells. Detectable levels of the cannabinoid receptor type 1 (CB1) were revealed in L6 cells. Exposure of differentiated L6 myotubes to the CB1 antagonist rimonabant (SR141716) selectively increased 2-deoxyglucose uptake (2-DG) in a time- and dose-dependent manner. A similar effect was induced by genetic silencing of CB1 by small interfering RNA. Protein expression profiling revealed that both the regulatory p85 and the catalytic p110 subunits of the phosphatidylinositol-3-kinase (PI3K) were increased by SR141716. No significant change in the cellular content of other known molecules regulating PI3K was observed. However, phosphoinositide-dependent kinase-1, Akt/protein kinase B, and protein kinase C activities were rapidly induced after SR141716 treatment of L6 cells in a PI3K-dependent manner. The stimulatory effect of SR141716 on PI3K expression and activity was largely prevented by N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), an inhibitor of the cAMP-dependent protein kinase. Moreover, SR141716-stimulated 2-DG uptake was blunted by the coincubation either with H-89 or with the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), both in L6 cells and in mouse primary myocytes. Thus, modulation of CB1 regulates glucose uptake at the level of the PI3K signaling system in skeletal muscle cells. Interfering with CB1 signaling may therefore ameliorate glucoregulatory functions in peripheral tissues.

[ARTICLES] Tryptophan Mutations at Azi-Etomidate Photo-Incorporation Sites on {alpha}1 or {beta}2 Subunits Enhance GABAA Receptor Gating and Reduce Etomidate Modulation

Stewart, D., Desai, R., Cheng, Q., Liu, A., Forman, S. A. — 2008-11-17

The potent general anesthetic etomidate produces its effects by enhancing GABA_A receptor activation. Its photolabel analog [³H]azi-etomidate labels residues within transmembrane domains on and β subunits: Met236 and βMet286. We hypothesized that these methionines contribute to etomidate sites formed at -β subunit interfaces and that increasing side-chain bulk and hydrophobicity at either locus would mimic etomidate binding and block etomidate effects. Channel activity was electrophysiologically quantified in ₁β₂_2L receptors with ₁M236W or β₂M286W mutations, in both the absence and the presence of etomidate. Measurements included spontaneous activation, GABA EC₅₀, etomidate agonist potentiation, etomidate direct activation, and rapid macrocurrent kinetics. Both ₁M236W and β₂M286W mutations induced spontaneous channel opening, lowered GABA EC₅₀, increased maximal GABA efficacy, and slowed current deactivation, mimicking effects of etomidate on ₁β₂_2L channels. These changes were larger with ₁M236W than with β₂M286W. Etomidate (3.2 µM) reduced GABA EC₅₀ much less in ₁M236Wβ₂_2L receptors (2-fold) than in wild type (23-fold). However, etomidate was more potent and efficacious in directly activating ₁M236Wβ₂_2L compared with wild type. In ₁β₂M286W_2L receptors, etomidate induced neither agonist-potentiation nor direct channel activation. These results support the hypothesis that ₁Met236 and β₂Met286 are within etomidate sites that allosterically link to channel gating. Although ₁M236W produced the larger impact on channel gating, β₂M286W produced more profound changes in etomidate sensitivity, suggesting a dominant role in drug binding. Furthermore, quantitative mechanistic analysis demonstrated that wild-type and mutant results are consistent with the presence of only one class of etomidate sites mediating both agonist potentiation and direct activation.

[ARTICLES] Potent Activation of Large-Conductance Ca2+-Activated K+ Channels by the Diphenylurea 1,3-Bis-[2-hydroxy-5-(trifluoromethyl)phenyl]urea (NS1643) in Pituitary Tumor (GH3) Cells

Wu, S.-N., Peng, H., Chen, B.-S., Wang, Y.-J., Wu, P.-Y., Lin, M.-W. — 2008-11-17

1,3-Bis-[2-hydroxy-5-(trifluoromethyl)phenyl]urea (NS1643) is reported to be an activator of human ether-à-go-go-related gene current. However, it remains unknown whether it has any effects on other types of ion channels. The effects of NS1643 on ion currents and membrane potential were investigated in this study. NS1643 stimulated Ca²⁺-activated K⁺ current [I_K(Ca)] in a concentration-dependent manner with an EC₅₀ value of 1.8 µM in pituitary tumor (GH₃) cells. In inside-out recordings, this compound applied to the intracellular side of the detached channels stimulated large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels with no change in single-channel conductance. It shifted the activation curve of BK_Ca channels to less depolarized voltages without altering the gating charge of the channels. NS1643-stimulated channel activity depended on intracellular Ca²⁺, and mean closed time during exposure to NS1643 was reduced. NS1643 (3 µM) had little or no effect on peak amplitude of ether-à-go-go-related gene-mediated K⁺ current evoked by membrane hyperpolarization, although it increased the amplitude of late-sustained components of K⁺ inward current, which was suppressed by paxilline but not by azimilide. NS1643 (3 µM) had no effect on L-type Ca²⁺ current. This compound reduced repetitive firing of action potentials, and further application of paxilline attenuated its decrease in firing rate. In addition, NS1643 enhanced BK_Ca-channel activity in human embryonic kidney 293T cells expressing -hSlo. In summary, we clearly show that NS1643 interacts directly with the BK_Ca channel to increase the amplitude of I_K(Ca) in pituitary tumor (GH₃) cells. The -subunit of the channel may be a target for the action of this small compound.

[ARTICLES] Inhibitors of GlyT1 Affect Glycine Transport via Discrete Binding Sites

2008-11-17

In the forebrain, synaptic glycine concentrations are regulated through the glycine transporter GlyT1. Because glycine is a coagonist of the N-methyl-d-aspartate (NMDA) receptor (NMDAR), which has been implicated in schizophrenia, inhibition of GlyT1 is thought to provide an option for the treatment of schizophrenia. In support of this hypothesis, GlyT1 inhibitors facilitate in vivo NMDAR function and demonstrate antipsychotic-like effects in animal models. Among the specific GlyT1 inhibitors, substituted N-methyl-glycine (sarcosine) derivatives (e.g., (R)-N[3-(4'fluorophenyl)-3-(4'phenyl-phenoxy)propyl]-sarcosine [NFPS], (R)-N[3-phenyl-3-(4'-(4-toluoyl)phenoxy)-propyl]sarcosine [(R)-NPTS], and (R,S)-(±)N-methyl-N-[(4-trifluoromethyl)phenoxy]-3-phenyl-propylglycine [Org24589]), and non-sarcosine-containing inhibitors, such as 2-chloro-N-[(S)-phenyl[(2S)-piperidin-2-yl] methyl]-3-trifluoromethyl benzamide, monohydrochloride (SSR504734), have been described. In the present study, we analyzed the mode of interaction of these compounds with GlyT1 by using electrophysiological measurements in Xenopus laevis oocytes, and with two binding assays, using [³H](R)-NPTS or 2-chloro-N-[(S)-phenyl[(2S)-N-methylpiperidin-2-yl]-methyl]-3-trifluoromethyl benzamide monohydrochloride ([³H]N-methyl-SSR504734) as radioligands. Inhibition of electrogenic glycine transport by sarcosine-based compounds was apparently irreversible and independent of glycine concentration. The latter indicates a noncompetitive mode of action. In contrast, both SSR504734 and N-methyl-SSR504734 exhibited reversible and competitive inhibition of glycine transport. In GlyT1-expressing membranes, the binding of the novel radioligand [³H]N-methyl-SSR504734 to a single site on GlyT1 was competitively displaced by glycine and SSR504734 but noncompetitively by sarcosine-based compounds. Inversely, [³H](R)-NPTS binding was competitively inhibited by sarcosine-based compounds, whereas glycine, SSR504734, and N-methyl-SSR504734 noncompetitively decreased maximal binding. Our data indicate that besides exerting an apparently irreversible or reversible inhibition, GlyT1 inhibitors differ by exhibiting either a noncompetitive or competitive mode of inhibition. The divergent modes of inhibition may significantly affect the efficacy and tolerability of these drugs.

[ARTICLES] The Selective Alzheimer's Disease Indicator-1 Gene (Seladin-1/DHCR24) Is a Liver X Receptor Target Gene

2008-11-17

The nuclear hormone receptors liver X receptor (LXR) and LXRβ function as physiological receptors for oxidized cholesterol metabolites (oxysterols) and regulate several aspects of cholesterol and lipid metabolism. Seladin-1 was originally identified as a gene whose expression was down-regulated in regions of the brain associated with Alzheimer's disease. Seladin-1 has been demonstrated to be neuroprotective and was later characterized as 3β-hydroxysterol-24 reductase (DHCR24), a key enzyme in the cholesterologenic pathway. Seladin-1 has also been shown to regulate lipid raft formation. In a whole genome screen for direct LXR target genes, we identified an LXR occupancy site within the second intron of the Seladin-1/DHCR24 gene. We characterized a novel LXR response element within the second intron of this gene that is able to confer LXR-specific ligand responsiveness to reporter gene in both HepG2 and human embryonic kidney 293 cells. Furthermore, we found that Seladin-1/DHCR24 gene expression is significantly decreased in skin isolated from LXRβ-null mice. Our data suggest that Seladin-1/DHCR24 is an LXR target gene and that LXR may regulate lipid raft formation.

[ARTICLES] Mechanisms of Myocyte Cytotoxicity Induced by the Multiple Receptor Tyrosine Kinase Inhibitor Sunitinib

Hasinoff, B. B., Patel, D., O'Hara, K. A. — 2008-11-17

The anticancer tyrosine kinase inhibitor sunitinib has been shown recently to be cardiotoxic. Using a neonatal rat myocyte model, we investigated various mechanisms that might be responsible for its cardiotoxicity. Sunitinib potently inhibited the enzyme activity of both AMP-activated protein kinase (AMPK) and the ribosomal S6 kinase RSK1 at therapeutically relevant concentrations. Heart tissue with its high energy needs might be particularly sensitive to inhibition of AMPK because of its role as an energy sensor regulating ATP levels. As measured by lactate dehydrogenase release, sunitinib treatment of myocytes caused dose-dependent damage at therapeutic levels. Sunitinib treatment also caused a dose-dependent reduction in myocyte protein levels of the phosphorylated and β isoforms of the AMPK phosphorylation target acetyl-Coenzyme A carboxylase. However, myocytes were not protected from sunitinib treatment by pretreating them with the AMPK-activating antidiabetic drug metformin. Sunitinib treatment of myocytes also did not affect cellular ATP levels. Together, these last two results do not suggest a major role for inhibition of AMPK in sunitinib-induced myocyte damage. Dexrazoxane, which is a clinically approved doxorubicin cardioprotective agent, also did not protect myocytes from damage, which suggests that sunitinib did not induce oxidative damage. In conclusion, even though sunitinib potently inhibits AMPK and RSK1, given the extreme lack of kinase selectivity that sunitinib exhibits, it is likely that inhibition of other kinases or combinations of kinases are responsible for the cardiotoxic effects of sunitinib.

[MINIREVIEW] Cardiovascular KCNQ (Kv7) Potassium Channels: Physiological Regulators and New Targets for Therapeutic Intervention

Mackie, A. R., Byron, K. L. — 2008-10-20

Potassium channels play an important role in electrical signaling of excitable cells such as neurons, cardiac myocytes, and vascular smooth muscle cells (VSMCs). In particular, the KCNQ (Kv7) family of voltage-activated K⁺ channels functions to stabilize negative resting membrane potentials and thereby opposes electrical excitability. Of the five known members of the mammalian Kv7 family, Kv7.1 was originally recognized for its role in cardiac myocytes, where it contributes to repolarization of the cardiac action potential. Kv7.2 to Kv7.5 were first discovered in neurons, in which they play a well characterized role in neurotransmitter-stimulated action potential firing. Over the past 5 years, important new roles for Kv7 channels have been identified. Kv7 channels have been found to be expressed in VSMCs from several vascular beds where they contribute to the regulation of vascular tone. There is evidence that Kv7.5 channels in VSMCs are targeted by the hormone vasopressin to mediate its physiological vasoconstrictor actions and evidence that neuronal Kv7 channels in the baroreceptors of the aortic arch adjust the sensitivity of the mechanosensitive neurons to changes in arterial blood pressure. These newly identified physiological roles for Kv7 channels in the cardiovascular system warrant increased attention because pharmacological modulators of this family of channels are being used clinically to treat a variety of neurological disorders. This raises questions about the cardiovascular side effects associated with existing therapies, but there is also obvious potential to capitalize on the established and evolving pharmacology of these channels to develop new therapies for cardiovascular diseases.

[PERSPECTIVE] Sensitization of Nociceptive Ion Channels by Inhaled Anesthetics--A Pain in the Gas?

Harrison, N., Nau, C. — 2008-10-20

A remarkable new article in this issue of Molecular Pharmacology (p. 1261) shows that the capsaicin-sensitive ion channel TRPV1 is sensitized to activation by chemical and physical stimuli in the presence of inhaled general anesthetics. This finding provides another example of an ion channel in which the anesthetic acts to modify channel gating. This may have important clinical implications in view of the role of TRPV1 in nociception.

[ARTICLES] Enhanced Anti-Inflammation of Inhaled Dexamethasone Palmitate Using Mannosylated Liposomes in an Endotoxin-Induced Lung Inflammation Model

2008-10-20

Inhalation of bacterial endotoxin induces pulmonary inflammation by activation of nuclear factor B (NFB), production of cytokines and chemokines, and neutrophil activation. Although glucocorticoids are the drugs of choice, administration of free drugs results in adverse effects as a result of a lack of selectivity for the inflammatory effector cells. Because alveolar macrophages play a key role in the inflammatory response in the lung, selective targeting of glucocorticoids to alveolar macrophages offers efficacious pharmacological intervention with minimal side effects. We have demonstrated previously the selective targeting of mannosylated liposomes to alveolar macrophages via mannose receptor-mediated endocytosis after intratracheal administration. In this study, the anti-inflammatory effects of dexamethasone palmitate incorporated in mannosylated liposomes (DPML) at 0.5 mg/kg via intratracheal administration were investigated in lipopolysaccharide-induced lung inflammation in rats. DPML significantly inhibited tumor necrosis factor , interleukin-1β, and cytokine-induced neutrophil chemoattractant-1 levels, suppressed neutrophil infiltration and myeloperoxidase activity, and inhibited NFB and p38 mitogen-activated protein kinase activation in the lung. These results prove the value of inhaled mannosylated liposomes as powerful targeting systems for the delivery of anti-inflammatory drugs to alveolar macrophages to improve their efficacy against lung inflammation.

[ARTICLES] Identification of a Putative Intracellular Allosteric Antagonist Binding-Site in the CXC Chemokine Receptors 1 and 2

2008-10-20

The chemokine receptors CXCR1 and CXCR2 are G-protein-coupled receptors (GPCRs) implicated in mediating cellular functions associated with the inflammatory response. Potent CXCR2 receptor antagonists have been discovered, some of which have recently entered clinical development. The aim of this study was to identify key amino acid residue differences between CXCR1 and CXCR2 that influence the relative antagonism by two compounds that have markedly different chemical structures. By investigating the effects of domain switching and point mutations, we found that the second extracellular loop, which contained significant amino acid sequence diversity, was not important for compound antagonism. We were surprised to find that switching the intracellular C-terminal 60 amino acid domains of CXCR1 and CXCR2 caused an apparent reversal of antagonism at these two receptors. Further investigation showed that a single amino acid residue, lysine 320 in CXCR2 and asparagine 311 in CXCR1, plays a predominant role in describing the relative antagonism of the two compounds. Homology modeling studies based on the structure of bovine rhodopsin indicated a potential intracellular antagonist binding pocket involving lysine 320. We conclude that residue 320 in CXCR2 forms part of a potential allosteric binding pocket on the intracellular side of the receptor, a site that is distal to the orthosteric site commonly assumed to be the location of antagonist binding to GPCRs. The existence of a common intracellular allosteric binding site at GPCRs related to CXCR2 may be of value in the design of novel antagonists for therapeutic intervention.

[ARTICLES] 1-[6-[[(17{beta})-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) Selectively Inhibits Kir3 and BK Channels in a Phospholipase C-Independent Fashion

Klose, A., Huth, T., Alzheimer, C. — 2008-10-20

1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) is widely used to inhibit phospholipase C (PLC)-mediated signaling, but we and others have also reported a PLC-independent block of Kir3 channels in native cells. To elaborate on this major side effect, we examined the action of U73122 and 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrollidinedione (U73343), a structurally related but not PLC-inhibiting analog, on Kir1.1, Kir2.1, or Kir3.1/3.2 channels expressed in HEK293 cells. Both compounds (10 µM) displayed an unusual degree of selectivity for Kir3, superior even to that of tertiapin, which discriminates between Kir3 and Kir2 but also inhibits Kir1.1. Recordings from mutant Kir2 and Kir3 channels showed that U73122 is unlikely to block Kir3 by interfering with binding of phosphatidylinositol 4,5-bisphosphate, and U73122 did not seem to act like a pore blocker. U73122 and U73343 also unexpectedly suppressed Ca²⁺-activated K⁺ channels of the large-conductance type (MaxiK, BK) in a PLC-independent fashion. In single-channel recordings, both compounds significantly decreased open probability of BK channels and slowed their ultrafast gating ("flickering") at very depolarized potentials. Alignment of the amino acid sequences of Kir3 and BK channels suggested that the highly selective effect of U73122/U73343 is mediated by a homologous domain within the long C-terminal ends. In fact, mutations in the C-terminal region of Kir2 and Kir3 channels significantly altered their sensitivity to the two compounds. Our data strongly caution against the use of U73122 when exploring signaling pathways involving Kir3 and BK channels. However, the apparent binding of U73122/U73343 to a common structural motif might be exploited to develop drugs selectively targeting Kir3 and BK channels.

[ARTICLES] Dynamic Effect of Bortezomib on Nuclear Factor-{kappa}B Activity and Gene Expression in Tumor Cells

2008-10-20

Nuclear factor-B (NF-B) influences the initiation, progression, and maintenance of diverse cancer types. Despite current therapeutic efforts to block hyperactive NF-B in cancer cells, the in vivo effects of a drug upon this complex pathway are unclear. We monitored NF-B activity and a fast-expressing reporter level simultaneously in head and neck squamous carcinoma cells by quantitative live microscopy. The real-time single cell assay revealed the tumor necrosis factor--induced oscillation of NF-B was echoed by equally dynamic reporter expression rate. Bortezomib is a proteasome inhibitor whose anticancer action is partly mediated through inhibition of NF-B. When administered to preactivated cells, the drug gave rise to distinct inhibition dynamics, with discrete pulses of reporter induction remaining for hours. These findings suggest that, contrary to a simplistic presumption for a pathway "blockade," the network dynamics and the intracellular pharmacokinetics of the inhibitor must be critically evaluated in developing strategies for optimal intervention of oncogenic pathways.

[ARTICLES] Differential Signaling Pathways in Angiotensin II- and Epidermal Growth Factor-stimulated Hepatic C9 Cells

Yin, X., Li, B., Chen, H., Catt, K. J. — 2008-10-20

Caveolin1 (Cav1) is an important component of the plasmamembrane microdomains, such as caveolae/lipid rafts, that are associated with angiotensin II type 1 (AT₁) and epidermal growth factor (EGF) receptors in certain cell types. The interactions of Cav1 with other signaling molecules that mediate AT₁ receptor function were analyzed in angiotensin II (Ang II)- and EGF-stimulated hepatic C9 cells. This study demonstrated that cholesterol-rich domains mediate the actions of early upstream signaling molecules such as Src and intracellular Ca²⁺ in cells stimulated by Ang II, but not by EGF, and that Cav1 has a scaffolding role in the process of mitogen-activated protein kinase activation. Furthermore, Cav1 phosphorylation by Ang II and EGF was regulated by intracellular Ca²⁺ and Src, further indicating reciprocal interactions among Cav1, Src, and intracellular Ca²⁺ through the AT₁ receptor. Phosphorylation of Cav1 and the EGF receptor by Ang II, but not of extracellular signal-regulated kinase 1/2, was dependent on intracellular Ca²⁺. The phosphatidylinositol 3-kinase inhibitors, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and wortmannin, differentially modulated both Cav1 and EGF receptor activation by Ang II through intracellular Ca²⁺. These findings further demonstrate the importance of Cav1 in conjunction with the receptor-mediated signaling pathways involved in cell proliferation and survival. It is clear that differential signaling pathways are operative in Ang II- and EGF-stimulated C9 cells and that cholesterol-enriched microdomains are essential components in cellular signaling processes that are dependent on specific agonists and/or cell types.

[ARTICLES] Reactive Oxygen Species Mediate p53 Activation and Apoptosis Induced by Sodium Nitroprusside in SH-SY5Y Cells

Cardaci, S., Filomeni, G., Rotilio, G., Ciriolo, M. R. — 2008-10-20

Sodium nitroprusside (SNP) is a water-soluble iron nitrosyl complex clinically used as a powerful vasodilator for treatment of hypertension; and, in basic research, it has been used to mainly investigate the cytotoxic effects of nitrosative stress. Although NO is considered a pharmacologically active molecule, not all of the biological effects of SNP are dependent on its NO moiety. To elucidate the molecular executioner(s) responsible for SNP cytotoxicity, this study determines the involvement of oxidative stress in p53 activation and apoptotic induction elicited by SNP in SH-SY5Y neuroblastoma cells. We demonstrate that proapoptotic activity of SNP is independent of NO production, because SNP and its 2-day light-exhausted compound SNP_ex trigger apoptosis to the same extent. We provide evidence for the occurrence of oxidative stress and oxidative damage during both SNP and SNP_ex exposure and demonstrate that iron-derived reactive oxygen species (ROS) are the genuine mediators of their cytotoxicity. We show that p53 is equally activated upon both SNP and SNP_ex treatments. Moreover, as demonstrated by small interfering RNA experiments, we indicate its primary role in the induction of apoptosis, suggesting the ineffectiveness of NO in its engagement. The attenuation of p53 levels, obtained by oxy-radical scavengers, is consistent with the recovery of cell viability and ROS decrease, demonstrate that SNP-mediated p53 activation is an event triggered by ROS and/or ROS-mediated damages. Together, our results suggest that investigations of the physiopathological effects of SNP should consider the role of ROS, other than NO, particularly in some conditions such as apoptotic induction and p53 activation.

[ARTICLES] Role of Key Transmembrane Residues in Agonist and Antagonist Actions at the Two Conformations of the Human {beta}1-Adrenoceptor

Baker, J. G., Proudman, R. G. W., Hawley, N. C., Fischer, P. M., Hill, S. J. — 2008-10-20

Studies with 4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP 12177) at the human β1-adrenoceptor have provided evidence for two binding modes or conformations that have markedly different pharmacological properties. Here, key transmembrane residues (Asp104, Asp138, Ser228, Ser229, Ser232, Phe341, Asn344 and Asn363) have been mutated to provide structural insights into the nature of these conformations. [³H]CGP 12177 binding and cAMP response element-mediated reporter gene studies confirmed that CGP 12177 was a neutral antagonist (log K_D = -9.18) at the "catecholamine site" and an agonist at the "CGP 12177 site" (log EC₅₀ = -8.12). Agonist responses to isoprenaline and CGP 12177 had different sensitivities to β1-antagonists (e.g., CGP 20712A; log K_D = -8.65 and -7.26, respectively). Site-directed mutagenesis showed that Asn363 and Asp138 were key residues for binding of agonists and antagonists, and they were also essential for the agonist actions of CGP 12177. S228A and S229A in transmembrane-spanning region (TM) 5 reduced the binding of CGP 12177 and had an identical effect on its agonist and antagonist actions. Both N344A and F341A in TM6 abolished the ability of CGP 20712A to discriminate between responses elicited by isoprenaline and CGP 12177. The fact that both Asp138 and Asn363 are absolutely required for CGP 12117 binding in both agonist and antagonist modes leads to the conclusion that the secondary agonist binding site for CGP 12117 must overlap with the catecholamine binding site. Modeling studies provide a basis for these overlapping sites with either the tert-butylamino group or the hydroxyethyloxy and imidazolone portions of CGP 12177 capable of forming polar interactions with Asp138 and Asn363.

[ARTICLES] General Anesthetics Sensitize the Capsaicin Receptor Transient Receptor Potential V1

Cornett, P. M., Matta, J. A., Ahern, G. P. — 2008-10-20

General anesthetics (GAs) are central nervous system depressants that render patients unresponsive to external stimuli. In contrast, many of these agents are also known to stimulate peripheral sensory nerves, raising the possibility that they may exacerbate tissue inflammation. We have found that pungent GAs excite sensory neurons by directly activating the transient receptor potential (TRP) A1 ion channel. Here, we show that GAs also sensitize the capsaicin receptor TRPV1, a key ion channel expressed in nociceptive neurons. Clinically relevant concentrations of isoflurane, sevoflurane, enflurane, and desflurane sensitize TRPV1 to capsaicin and protons and reduce the threshold for heat activation. Furthermore, isoflurane directly activates TRPV1 after stimulation of protein kinase C. Likewise, isoflurane excites TRPV1 and sensory neurons during concomitant application of bradykinin, a key inflammatory mediator formed during tissue injury. Thus, GAs can enhance the activation of TRPV1 that occurs during surgically induced tissue damage. These results support the hypothesis that some GAs, through direct actions at TRP channels, increase postsurgical pain and inflammation.

[ARTICLES] Ligand-Mediated Regulation of Peroxisome Proliferator-Activated Receptor (PPAR) {beta}/{delta}: A Comparative Analysis of PPAR-Selective Agonists and All-trans Retinoic Acid

Rieck, M., Meissner, W., Ries, S., Muller-Brusselbach, S., Muller, R. — 2008-10-20

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that modulate target gene expression in response to natural fatty acid ligands and synthetic agonists. It is noteworthy that all trans-retinoic acid (atRA) has recently been reported to act as a ligand for PPARβ/, to activate its transcriptional activity, and, in contrast to the "classic" function of atRA, to stimulate cell proliferation (Schug et al., 2007). Here, we report that in contrast to synthetic PPARβ/ agonists, atRA failed to induce the transcriptional activity of PPARβ/ using different types of reporter gene assays. Likewise, atRA did not affect the expression of the bona fide PPARβ/ target genes ADRP and ANGPTL4 but strongly increased expression of the retinoic acid target gene CYP26A under the identical experimental conditions. Consistent with these observations, atRA did not compete with established PPARβ/ agonists in a ligand binding assay, and atRA did not enable the interaction of PPARβ/ with a coactivator peptide in a time-resolved fluorescence resonance energy transfer assay in vitro. These results are in sharp contrast to the effect of established PPARβ/ agonists in both in vitro assays. Taken as a whole, these data strongly suggest that atRA does not function as a ligand of PPARβ/ in any of the experimental systems tested and that the previously reported atRA effects are more likely to reflect an uncharacterized and less direct mechanism.

[ARTICLES] Morphine Desensitization, Internalization, and Down-Regulation of the {micro} Opioid Receptor Is Facilitated by Serotonin 5-Hydroxytryptamine2A Receptor Coactivation

Lopez-Gimenez, J. F., Vilaro, M. T., Milligan, G. — 2008-10-20

Analysis of the distribution of mRNA encoding the serotonin (5-hydroxytryptamine) 5-HT_2A receptor and the µ opioid peptide receptor in rat brain demonstrated their coexpression in neurons in several distinct regions. These regions included the periaqueductal gray, an area that plays an important role in morphine-induced analgesia but also in the development of tolerance to morphine. To explore potential cross-regulation between these G protein-coupled receptors, the human µ opioid peptide receptor was expressed stably and constitutively in Flp-In T-REx human embryonic kidney 293 cells that harbored the human 5-HT_2A receptor at the inducible Flp-In locus. In the absence of the 5-HT_2A receptor, pretreatment with the enkephalin agonist [d-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin but not with the alkaloid agonist morphine produced desensitization, internalization, and down-regulation of the µ opioid peptide receptor. Induction of 5-HT_2A receptor expression in these cells resulted in up-regulation of µ opioid peptide receptor levels that was blocked by both a 5-HT_2A receptor inverse agonist and selective inhibition of signaling via G_q/G₁₁ G proteins. After induction of the 5-HT_2A receptor, coaddition of 5-HT with morphine now also resulted in desensitization, receptor internalization, and down-regulation of the µ opioid peptide receptor. It has been argued that enhancement of µ opioid peptide receptor internalization in response to morphine would limit the development of tolerance without limiting analgesia. These data suggest that selective activation of the 5-HT_2A receptor in concert with treatment with morphine might achieve this aim.

[ARTICLES] Mitochondria-Dependent Reactive Oxygen Species-Mediated Programmed Cell Death Induced by 3,3'-Diindolylmethane through Inhibition of F0F1-ATP Synthase in Unicellular Protozoan Parasite Leishmania donovani

2008-10-20

Mitochondria are the principal site for the generation of cellular ATP by oxidative phosphorylation. F0F1-ATP synthase, a complex V of the electron transport chain, is an important constituent of mitochondria-dependent signaling pathways involved in apoptosis. In the present study, we have shown for the first time that 3,3'-diindolylmethane (DIM), a DNA topoisomerase I poison, inhibits mitochondrial F0F1-ATP synthase of Leishmania donovani and induces programmed cell death (PCD), which is a novel insight into the mechanism in protozoan parasites. DIM-induced inhibition of F0F1-ATP synthase activity causes depletion of mitochondrial ATP levels and significant stimulation of mitochondrial reactive oxygen species (ROS) production, followed by depolarization of mitochondrial membrane potential (_m). Because _m is the driving force for mitochondrial ATP synthesis, loss of _m results in depletion of cellular ATP level. The loss of _m causes the cellular ROS generation and in turn leads to the oxidative DNA lesions followed by DNA fragmentation. In contrast, loss of _m leads to release of cytochrome c into the cytosol and subsequently activates the caspase-like proteases, which lead to oligonucleosomal DNA cleavage. We have also shown that mitochondrial DNA-depleted cells are insensitive to DIM to induce PCD. Therefore, mitochondria are necessary for cytotoxicity of DIM in kinetoplastid parasites. Taken together, our study indicates for the first time that DIM-induced mitochondrial dysfunction by inhibition of F0F1-ATP synthase activity leads to PCD in Leishmania spp. parasites, which could be exploited to develop newer potential therapeutic targets.

[ARTICLES] Full Pharmacological Efficacy of a Novel S1P1 Agonist That Does Not Require S1P-Like Headgroup Interactions

2008-10-20

Strong evidence exists for interactions of zwitterionic phosphate and amine groups in sphingosine-1 phosphate (S1P) to conserved Arg and Glu residues present at the extracellular face of the third transmembrane domain of S1P receptors. The contribution of Arg¹²⁰ and Glu¹²¹ for high-affinity ligand-receptor interactions is essential, because single-point R¹²⁰A or E¹²¹A S1P₁ mutants neither bind S1P nor transduce S1P function. Because S1P receptors are therapeutically interesting, identifying potent selective agonists with different binding modes and in vivo efficacy is of pharmacological importance. Here we describe a modestly water-soluble highly selective S1P₁ agonist [2-(4-(5-(3,4-diethoxyphenyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl amino) ethanol (CYM-5442)] that does not require Arg¹²⁰ or Glu¹²¹ residues for activating S1P₁-dependent p42/p44 mitogen-activated protein kinase phosphorylation, which defines a new hydrophobic pocket in S1P₁. CYM-5442 is a full agonist in vitro for S1P₁ internalization, phosphorylation, and ubiquitination. It is noteworthy that CYM-5442 was a full agonist for induction and maintenance of S1P₁-dependent blood lymphopenia, decreasing B lymphocytes by 65% and T lymphocytes by 85% of vehicle. Induction of CYM-5442 lymphopenia was dose- and time-dependent, requiring serum concentrations in the 50 nM range. In vitro measures of S1P₁ activation by CYM-5442 were noncompetitively inhibited by a specific S1P₁ antagonist [(R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146)], competitive for S1P, 2-amino-2-(4-octylphenethyl)propane-1,3-diol (FTY720-P), and 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2, 4-oxadiazole (SEW2871). In addition, lymphopenia induced by CYM-5442 was reversed by W146 administration or upon pharmacokinetic agonist clearance. Pharmacokinetics in mice also indicated that CYM-5442 partitions significantly in central nervous tissue. These data show that CYM-5442 activates S1P₁-dependent pathways in vitro and to levels of full efficacy in vivo through a hydrophobic pocket separate from the orthosteric site of S1P binding that is headgroup-dependent.

[ARTICLES] Mitochondrial Respiratory Enzyme Complexes in Rostral Ventrolateral Medulla as Cellular Targets of Nitric Oxide and Superoxide Interaction in the Antagonism of Antihypertensive Action of eNOS Transgene

Kung, L.-C., Chan, S. H. H., Wu, K. L. H., Ou, C.-C., Tai, M.-H., Chan, J. Y. H. — 2008-10-20

Overproduction of nitric oxide (NO) by gene transduction of endothelial NO synthase (eNOS) in rostral ventrolateral medulla (RVLM), which is responsible for maintenance of vasomotor tone, reduces arterial pressure in spontaneously hypertensive rats (SHR). This NO-induced vasodepression, however, is not sustained and is followed by rebound hypertension. Because superoxide anion (O₂^.) level is increased and synthesis or activity of mitochondrial manganese superoxide dismutase (SOD2) is reduced in RVLM during hypertension, we hypothesized that an interaction between NO and O₂^. in RVLM, using mitochondrial respiratory enzyme complexes (MRC) as the cellular target, contributes to those cardiovascular outcomes after eNOS gene transduction in SHR. The present study assessed this hypothesis using adenoviral vectors to overexpress eNOS (AdeNOS) and/or SOD2 (AdSOD2) in RVLM of SHR or normotensive Wistar-Kyoto (WKY) rats. Microinjection of AdeNOS bilaterally into RVLM elicited 35% depression of MRC-I enzyme activity and evoked 60% and 50% increase in O₂^. and peroxynitrite level in RVLM of SHR, but not WKY rats, which was reversed by cotransduced AdSOD2 or treatment with peroxynitrite decomposition catalyst. Cotransduction of AdeNOS and AdSOD2 in RVLM of SHR elicited significantly greater decreases in arterial pressure and heart rate than those promoted by the individual transgene and prevented the AdeNOS-induced rebound hypertension. We conclude that an interactive action between NO and O₂^. on MRC-I in RVLM via formation of peroxynitrite contributes to the unsustained hypotensive effects of NO after overexpression of eNOS in SHR. The mitochondria-derived O₂^. also mediates the rebound hypertension induced by eNOS transgene in RVLM of SHR.

[ARTICLES] Coassembly of Different Sulfonylurea Receptor Subtypes Extends the Phenotypic Diversity of ATP-sensitive Potassium (KATP) Channels

2008-10-20

K_ATP channels are metabolic sensors and targets of potassium channel openers (KCO; e.g., diazoxide and pinacidil). They comprise four sulfonylurea receptors (SUR) and four potassium channel subunits (Kir6) and are critical in regulating insulin secretion. Different SUR subtypes (SUR1, SUR2A, SUR2B) largely determine the metabolic sensitivities and the pharmacological profiles of K_ATP channels. SUR1- but not SUR2-containing channels are highly sensitive to metabolic inhibition and diazoxide, whereas SUR2 channels are sensitive to pinacidil. It is generally believed that SUR1 and SUR2 are incompatible in channel coassembly. We used triple tandems, T1 and T2, each containing one SUR (SUR1 or SUR2A) and two Kir6.226 (last 26 residues are deleted) to examine the coassembly of different SUR. When T1 or T2 was expressed in Xenopus laevis oocytes, small whole-cell currents were activated by metabolic inhibition (induced by azide) plus a KCO (diazoxide for T1, pinacidil for T2). When coexpressed with any SUR subtype, the activated-currents were increased by 2- to 13-fold, indicating that different SUR can coassemble. Consistent with this, heteromeric SUR1+SUR2A channels were sensitive to azide, diazoxide, and pinacidil, and their single-channel burst duration was 2-fold longer than that of the T1 channels. Furthermore, SUR2A was coprecipitated with SUR1. Using whole-cell recording and immunostaining, heteromeric channels could also be detected when T1 and SUR2A were coexpressed in mammalian cells. Finally, the response of the SUR1+SUR2A channels to azide was found to be intermediate to those of the homomeric channels. Therefore, different SUR subtypes can coassemble into K_ATP channels with distinct metabolic sensitivities and pharmacological profiles.

[ARTICLES] Discovery, Characterization, and Antiparkinsonian Effect of Novel Positive Allosteric Modulators of Metabotropic Glutamate Receptor 4

2008-10-20

Parkinson's disease (PD) is caused by the death of dopamine neurons in the basal ganglia and results in motor symptoms such as tremor and bradykinesia. Activation of metabotropic glutamate receptor 4 (mGluR4) has been shown to modulate neurotransmission in the basal ganglia and results in antiparkinsonian effects in rodent PD models. N-Phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) is a positive allosteric modulator (PAM) of mGluR4 that has been used to further validate the role of mGluR4 in PD, but the compound suffers from a lack of selectivity, relatively low potency, and poor solubility. Via high-throughput screening, we discovered more than 400 novel PAMs of mGluR4. Compounds derived from a novel chemical scaffold were characterized in vitro at both rat and human mGluR4 using two distinct assays of mGluR4 function. The lead compound was approximately 8-fold more potent than PHCCC, enhanced the potency of glutamate at mGluR4 by 8-fold, and did not show any significant potentiator or antagonist activity at other mGluR subtypes. Resolution of the regioisomers of the lead revealed that the cis regioisomer, (±)-cis-2-(3,5-dichlorphenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041), contained the majority of the mGluR4 PAM activity and also exhibited partial agonist activity at mGluR4 at a site that was distinct from the glutamate binding site, suggesting that this compound is a mixed allosteric agonist/PAM of mGluR4. VU0155041 was soluble in an aqueous vehicle, and intracerebroventricular administration of 31 to 316 nmol of VU0155041 dose-dependently decreased haloperidol-induced catalepsy and reserpine-induced akinesia in rats. These exciting results provide continued support for mGluR4 as a therapeutic target in PD.

[ARTICLES] Interleukin-8 Expression Is Regulated by Histone Deacetylases through the Nuclear Factor-{kappa}B Pathway in Breast Cancer

2008-10-20

We have reported recently that the chemokine interleukin 8 (IL-8)/CXCL8 was overexpressed in invasive estrogen receptor (ER)-negative breast cancer cells compared with ER-positive breast cancer cells. We now demonstrate that histone deacetylases (HDACs) play an essential role in the regulation of IL-8 gene expression in ER-positive MCF-7 breast cancer cells. Treatment of MCF-7 cells with the HDAC inhibitor trichostatin A (TSA) led to a strong up-regulation of IL-8 protein and RNA levels in MCF-7 cells. The up-regulation of IL-8 in MCF-7 cells was time- and concentration-dependent. Moreover, run-on and transfection experiments demonstrated that IL-8 induction by HDAC inhibitors was transcriptional and involved mainly the nuclear factor-B (NF-B) site of the IL-8 promoter. These observations are corroborated by an up-regulation of NF-B activity in MCF-7 cells in the presence of TSA. In addition, blocking NF-B pathway by adenoviral delivery of a dominant-negative IBorIB kinase complex 2 (IKK2) mutant abolished IL-8 gene induction by histone deacetylase inhibitors. HDAC inhibitors triggered IKK phosphorylation and up-regulated p65 nuclear translocation, although they decreased the protein levels of IB, which accounts for NF-B activation. TSA increased binding of acetylated histone 3 to the IL-8 gene promoter. In summary, our results demonstrate that NF-B pathway repression by HDAC is responsible for the low expression of IL-8 in ER-positive breast cancer cells.

[ARTICLES] A Hypomorphic Allele of Aryl Hydrocarbon Receptor-Associated Protein-9 Produces a Phenocopy of the Ahr-Null Mouse

Lin, B. C., Nguyen, L. P., Walisser, J. A., Bradfield, C. A. — 2008-10-20

The aryl hydrocarbon receptor-associated protein-9 (ARA9) is a chaperone of the aryl hydrocarbon receptor (AHR). The AHR has been shown to play a late developmental role in the normal closure of a fetal hepatovascular shunt known as the ductus venosus (DV). Given that Ara9-null mice display early embryonic lethality, we generated a hypomorphic Ara9 allele (designated Ara9^fxneo) that displays reduced ARA9 protein expression. In an effort to demonstrate the role of ARA9 protein in AHR-mediated DV closure, we used combinations of Ara9 wild-type [Ara9(+/+)], null [Ara9(-/-)], and hypomorphic [Ara9(fxneo/fxneo)] alleles to produce mice with a graded expression of the ARA9 protein. Liver perfusion studies demonstrated that although none of the Ara9(+/+) mice displayed a patent DV, the shunt was observed in 10% of the Ara9(+/fxneo) mice, 55% of the Ara9(+/-) mice, and 83% of the Ara9(fxneo/fxneo) mice. That expression level of ARA9 correlates with the frequency of a phenocopy of the Ahr-null allele supports the conclusion that the ARA9 protein is essential for AHR signaling during development.