Molecular Pharmacology current issue

[PERSPECTIVE] Seven Transmembrane Receptors as Nature's Prototype Allosteric Protein: De-emphasizing the Geography of Binding

Kenakin, T. P. — 2008-08-21

The article in this issue by Redka et al. (p. 834) illustrates some interesting interactions between classified orthosteric (bind to the same recognition site as endogenous agonist) and allosteric (bind to a different site) ligands. Of particular interest are the methods used to deal with an obfuscating factor in these kinds of studies, namely the propensity of seven transmembrane receptors to form dimers and thus demonstrate allosteric effects through binding at the orthosteric site. The judicious use of kinetics to detect and quantify allosteric action also is demonstrated. The various unique properties of allosteric modulators are discussed in the context of the increasing prevalence of allosteric ligands as investigational drugs.

[ACCELERATED COMMUNICATION] Ligand-Dependent Oligomerization of Dopamine D2 and Adenosine A2A Receptors in Living Neuronal Cells

Vidi, P.-A., Chemel, B. R., Hu, C.-D., Watts, V. J. — 2008-08-21

Adenosine A_2A and dopamine D₂ receptors (A_2A and D₂) associate in homo- and heteromeric complexes in the striatum, providing a structural basis for their mutual antagonism. At the cellular level, the portion of receptors engaging in homo- and heteromers, as well as the effect of persistent receptor activation or antagonism on the cell oligomer repertoire, are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to visualize A_2A and D₂ oligomerization in the Cath.a differentiated neuronal cell model. Receptor fusions to BiFC fluorescent protein fragments retained their function when expressed alone or in A_2A/A_2A, D₂/D₂, and A_2A/D₂ BiFC pairs. Robust fluorescence complementation reflecting A_2A/D₂ heteromers was detected at the cell membrane as well as in endosomes. In contrast, weaker BiFC signals, largely confined to intracellular domains, were detected with A_2A/dopamine D₁ BiFC pairs. Multicolor BiFC was used to simultaneously visualize A_2A and D₂ homo- and heteromers in living cells and to examine drug-induced changes in receptor oligomers. Prolonged D₂ stimulation with quinpirole lead to the internalization of D₂/D₂ and A_2A/D₂ oligomers and resulted in decreased A_2A/D₂ relative to A_2A/A_2A oligomer formation. Opposing effects were observed in cells treated with D₂ antagonists or with the A_2A agonist 5'-N-methylcarboxamidoadenosine (MECA). Subsequent radioreceptor binding analysis indicated that the drug-induced changes in oligomer formation were not readily explained by alterations in receptor density. These observations support the hypothesis that long-term drug exposure differentially alters A_2A/D₂ receptor oligomerization and provide the first demonstration for the use of BiFC to monitor drug-modulated GPCR oligomerization.

[ARTICLES] Mutational Analysis of the Conserved Asp2.50 and ERY Motif Reveals Signaling Bias of the Urotensin II Receptor

2008-08-21

Class A (rhodopsin-like) G protein-coupled receptors possess conserved residues and motifs that are important for their specific activity. In the present study, we examined the role of residue Asp97^2.50 as well as residues Glu147^3.49, Arg148^3.50, and Tyr149^3.51 of the ERY motif on the functionality of the urotensin II receptor (UT). Mutations D97^2.50A, R148^3.50A, and R148^3.50H abolished the ability of UT to activate phospholipase C, whereas mutations E147^3.49A and Y149^3.51A reduced the ability to activate PLC by 50%. None of the mutants exhibited constitutive activity. However, R148^3.50A and R148^3.50H promoted ERK1/2 activation, which was abolished by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase activity. Both these mutants were capable of directly activating EGFR, which confirmed that they activated the mitogen-activated protein kinase (MAPK) pathway by a G_q/11-independent transactivation of EGFR. The D97^2.50A, R148^3.50A, and R148^3.50H mutants did not readily internalize and did not promote translocation or colocalize with β-arrestin2-GFP. Finally, the agonist-induced internalization of the E147^3.49A mutant receptor was significantly increased compared with wild-type receptor. This study highlights the major contribution of the conserved Asp^2.50 residue to the functionality of the UT receptor. The Arg residue in the ERY motif of UT is an important structural element in signaling crossroads that determine whether G_q/11-dependent and -independent events can occur.

[ARTICLES] Different Internalization Properties of the {alpha}1a- and {alpha}1b-Adrenergic Receptor Subtypes: The Potential Role of Receptor Interaction with {beta}-Arrestins and AP50

Stanasila, L., Abuin, L., Dey, J., Cotecchia, S. — 2008-08-21

The internalization properties of the 1a- and 1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the 1b-AR displayed robust agonist-induced endocytosis, the 1a-AR did not. Constitutive internalization of the 1a-AR was negligible, whereas the 1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the 1-AR subtypes with β-arrestins 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both coimmunoprecipitation experiments and β-arrestin translocation assays indicated that the agonistinduced interaction of the 1a-AR with β-arrestins was much weaker than that of the 1b-AR. In addition, the 1a-AR did not bind AP50. The 1b-AR mutant M8, lacking the main phosphorylation sites in the receptor C tail, was unable to undergo endocytosis and was profoundly impaired in binding β-arrestins despite its binding to AP50. In contrast, the 1b-AR mutant R8, lacking AP50 binding, bound β-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that β-arrestin 2 plays a prominent role in 1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the 1a- and 1b-AR and provide evidence that the lack of significant endocytosis of the 1a-AR is linked to its poor interaction with β-arrestins as well as with AP50. We also provide evidence that the integrity of the phosphorylation sites in the C tail of the 1b-AR is important for receptor/β-arrestin interaction and that this interaction is the main event triggering receptor internalization.

[ARTICLES] Effects of Active and Inactive Phospholipase D2 on Signal Transduction, Adhesion, Migration, Invasion, and Metastasis in EL4 Lymphoma Cells

2008-08-21

The phosphatidylcholine-using phospholipase D (PLD) isoform PLD2 is widely expressed in mammalian cells and is activated in response to a variety of promitogenic agonists. In this study, active and inactive hemagglutinin-tagged human PLD2 (HA-PLD2) constructs were stably expressed in an EL4 cell line lacking detectable endogenous PLD1 or PLD2. The overall goal of the study was to examine the roles of PLD2 in cellular signal transduction and cell phenotype. HA-PLD2 confers PLD activity that is activated by phorbol ester, ionomycin, and okadaic acid. Proliferation and Erk activation are unchanged in cells transfected with active PLD2; proliferation rate is decreased in cells expressing inactive PLD2. Basal tyrosine phosphorylation of focal adhesion kinase (FAK) is increased in cells expressing active PLD2, as is phosphorylation of Akt; inactive PLD2 has no effect. Expression of active PLD2 is associated with increased spreading and elongation of cells on tissue culture plastic, whereas inactive PLD2 inhibits cell spreading. Inactive PLD2 also inhibits cell adhesion, migration, and serum-induced invasion. Cells expressing active PLD2 form metastases in syngeneic mice, as do the parental cells; cells expressing inactive PLD2 form fewer metastases than parental cells. In summary, active PLD2 enhances FAK phosphorylation, Akt activation, and cell invasion in EL4 lymphoma cells, whereas inactive PLD2 exerts inhibitory effects on adhesion, migration, invasion, and tumor formation. Overall, expression of active PLD2 enhances processes favorable to lymphoma cell metastasis, whereas expression of inactive PLD2 inhibits metastasis.

[ARTICLES] Pharmacological Characterization of Membrane-Expressed Human Trace Amine-Associated Receptor 1 (TAAR1) by a Bioluminescence Resonance Energy Transfer cAMP Biosensor

2008-08-21

Trace amines are neurotransmitters whose role in regulating invertebrate physiology has been appreciated for many decades. Recent studies indicate that trace amines may also play a role in mammalian physiology by binding to a novel family of G protein-coupled receptors (GPCRs) that are found throughout the central nervous system. A major obstacle impeding the careful pharmacological characterization of trace amine associated receptors (TAARs) is their extremely poor membrane expression in model cell systems, and a molecular basis for this phenomenon has not been determined. In the present study, we show that the addition of an asparagine-linked glycosylation site to the N terminus of the human trace amine associated receptor 1 (TAAR1) is sufficient to enable its plasma membrane expression, and thus its pharmacological characterization with a novel cAMP EPAC (exchange protein directly activated by cAMP) protein based bioluminescence resonance energy transfer (BRET) biosensor. We applied this novel cAMP BRET biosensor to evaluate the activity of putative TAAR1 ligands. This study represents the first comprehensive investigation of the membrane-expressed human TAAR1 pharmacology. Our strategy to express TAARs and to identify their ligands using a cAMP BRET assay could provide a foundation for characterizing the functional role of trace amines in vivo and suggests a strategy to apply to groups of poorly expressing GPCRs that have remained difficult to investigate in model systems.

[ARTICLES] Midazolam Reverses Salicylate-Induced Changes in Brain-Derived Neurotrophic Factor and Arg3.1 Expression: Implications for Tinnitus Perception and Auditory Plasticity

2008-08-21

Tinnitus is a phantom auditory perception, which can be induced via application of concentrated sodium salicylate, and is known to be associated with hearing loss and altered neuronal excitability in peripheral and central auditory neurons. The molecular features of this excitability, however, has been poorly characterized to date. Brain-derived neurotrophic factor (BDNF), the activity-dependent cytoskeletal protein (Arg3.1, also known as Arc), and c-Fos are known to be affected by changes in excitability and plasticity. Using reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemistry, the expression of these genes was monitored in the rat auditory system after local (cochlear) and systemic application of salicylate. Induction of tinnitus and hearing loss was verified in a behavioral model. Regardless of the mode of salicylate application, a common pattern became evident: 1) BDNF mRNA expression was increased in the spiral ganglion neurons of the cochlea; and 2) Arg3.1 expression was significantly reduced in the auditory cortex. Local application of the GABA_A receptor modulator midazolam resulted in the reversal not only of salicylate-induced changes in cochlear BDNF expression, but also in cortical Arg3.1 expression, indicating that the tinnitus-associated changes in cochlear BDNF expression trigger the decline of cortical Arg3.1 expression. Furthermore, local midazolam application reduced tinnitus perception in the animal model. These findings support Arg3.1 and BDNF as markers for activity changes in the auditory system and suggest a role of GABAergic inhibition of cochlear neurons in the modulation of Arg3.1 plasticity changes in the auditory cortex and tinnitus perception.

[ARTICLES] Ligand-Receptor Interactions at the Parathyroid Hormone Receptors: Subtype Binding Selectivity Is Mediated via an Interaction between Residue 23 on the Ligand and Residue 41 on the Receptor

Mann, R., Wigglesworth, M. J., Donnelly, D. — 2008-08-21

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) bind and activate the PTH/PTHrP receptor (PTH-1R). However, while the related receptor PTH-2R responds potently to PTH, it is not activated by PTHrP. Two hormone sites are known to be responsible for these different potencies. First, the absence of efficacy for PTHrP at PTH-2R is due to the presence of His-5 in PTHrP (Ile-5 in PTH), which interacts with the receptor's juxtamembrane domain. Second, PTHrP has lower affinity than PTH for PTH-2R because of the presence of Phe-23 (Trp-23 in PTH), which interacts with the receptor's N-terminal extracellular domain. We used these different receptor subtype properties to demonstrate that residue 41 in PTH-1R, when either the native Leu or substituted by Ile or Met, can accommodate either Phe or Trp at position 23 of the ligand. However, when Leu-41 is substituted by a smaller side chain, either Ala or Val (its equivalent residue in PTH-2R), the receptor becomes highly selective for those peptide ligands with Trp-23. Hence, despite the conservative nature of the substitutions found in the native ligands (Phe for Trp) and receptors (Leu for Val), they nevertheless enable a significant degree of selectivity to be achieved. Analysis of this functionally important ligand-receptor contact, within the context of the recent X-ray structure of the peptide-bound PTH-1R N domain, reveals the nature of the selectivity filter and how it is by-passed in PTH-1R.

[ARTICLES] Mutations of the GABA-A Receptor {alpha}1 Subunit M1 Domain Reveal Unexpected Complexity for Modulation by Neuroactive Steroids

2008-08-21

Neuroactive steroids are among the most efficacious modulators of the mammalian GABA-A receptor. Previous work has proposed that receptor potentiation is mediated by steroid interactions with a site defined by the residues 1Asn407/Tyr410 in the M4 transmembrane domain and residue 1Gln241 in the M1 domain. We examined the role of residues in the 1 subunit M1 domain in the modulation of the rat 1β22L GABA-A receptor by neuroactive steroids. The data demonstrate that the region is critical to the actions of potentiating neuroactive steroids. Receptors containing the 1Q241W or 1Q241L mutations were insensitive to (3,5)-3-hydroxypregnan-20-one (35P), albeit with different underlying mechanisms. The 1Q241S mutant was potentiated by 35P, but the kinetic mode of potentiation was altered by the mutation. It is noteworthy that the 1Q241L mutation had no effect on channel potentiation by (3,5)-3-hydroxymethyl-pregnan-20-one, but mutation of the neighboring residue, 1Ser240, prevented channel modulation. A steroid lacking an H-bonding group on C3 (5-pregnan-20-one) potentiated the wild-type receptor but not the 1Q241L mutant. The findings are consistent with a model in which the 1Ser240 and 1Gln241 residues shape the surface to which steroid molecules bind.

[ARTICLES] Ethanol Modulates BKCa Channels by Acting as an Adjuvant of Calcium

Liu, J., Vaithianathan, T., Manivannan, K., Parrill, A., Dopico, A. M. — 2008-08-21

Ethanol modulation of calcium- and voltage-gated potassium (slo1) channels alters neuronal excitability, cerebrovascular tone, brain function, and behavior, yet the mechanism of this modulation remains unknown. Using patch-clamp electrophysiology on recombinant BK_Ca channels cloned from mouse brain and expressed in Xenopus laevis oocytes, we demonstrate that ethanol, even at concentrations maximally effective to modulate BK_Ca channel function (100 mM), fails to gate the channel in absence of activating calcium. Moreover, ethanol does not modify intrinsic, voltage- or physiological magnesium-driven gating. The alcohol works as an adjuvant of calcium by selectively facilitating calcium-driven gating. This facilitation, however, renders differential ethanol effects on channel activity: potentiation at low (<10 µM) and inhibition at high (>10 µM) calcium, this dual pattern remaining largely unmodified by coexpression of brain slo1 channels with the neuronally abundant BK_Ca channel β₄ subunit. Calcium recognition by either of the slo1 high-affinity sensors (calcium bowl and RCK1 Asp362/Asp367) is required for ethanol to amplify channel activation by calcium. The Asp362/Asp367 site, however, is necessary and sufficient to sustain ethanol inhibition. This inhibition also results from ethanol facilitation of calcium action; in this case, ethanol favors channel dwelling in a calcium-driven, low-activity mode. The agonist-adjuvant mechanism that we advance from the calcium-ethanol interaction on slo1 might be applicable to data of ethanol action on a wide variety of ligand-gated channels.

[ARTICLES] A Peptide Accelerating the Conversion of Plasminogen Activator Inhibitor-1 to an Inactive Latent State

2008-08-21

The serpin plasminogen activator inhibitor-1 (PAI-1) is a specific inhibitor of plasminogen activators and a potential therapeutic target in cancer and cardiovascular diseases. Accordingly, formation of a basis for development of specific PAI-1-inactivating agents is of great interest. One possible inactivation mode for PAI-1 is conversion to the inactive, so-called latent state. We have now screened a phage-displayed peptide library with PAI-1 as bait and isolated a 31-residue cysteine-rich peptide that will be referred to as paionin-4. A recombinant protein consisting of paionin-4 fused to domains 1 and 2 of the phage coat protein g3p caused a 2- to 3-fold increase in the rate of spontaneous inactivation of PAI-1. Paionin-4-D1D2 bound PAI-1 with a K_D in the high nanomolar range. Using several biochemical and biophysical methods, we demonstrate that paionin-4-D1D2-stimulated inactivation consists of an acceleration of conversion to the latent state. As demonstrated by site-directed mutagenesis and competition with other PAI-1 ligands, the binding site for paionin-4 was localized in the loop between -helix D and β-strand 2A. We also demonstrate that a latency-inducing monoclonal antibody has an overlapping, but not identical binding site, and accelerates latency transition by another mechanism. Our results show that paionin-4 inactivates PAI-1 by a mechanism clearly different from other peptides, small organochemical compounds, or antibodies, whether they cause inactivation by stimulating latency transition or by other mechanisms, and that the loop between -helix D and β-strand 2A can be a target for PAI-1 inactivation by different types of compounds.

[ARTICLES] Inhibition of I{kappa}B Kinase-Nuclear Factor-{kappa}B Signaling Pathway by 3,5-Bis(2-flurobenzylidene)piperidin-4-one (EF24), a Novel Monoketone Analog of Curcumin

2008-08-21

The nuclear factor-B (NF-B) signaling pathway has been targeted for therapeutic applications in a variety of human diseases, includuing cancer. Many naturally occurring substances, including curcumin, have been investigated for their actions on the NF-B pathway because of their significant therapeutic potential and safety profile. A synthetic monoketone compound termed 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24) was developed from curcumin and exhibited potent anticancer activity. Here, we report a mechanism by which EF24 potently suppresses the NF-B signaling pathway through direct action on IB kinase (IKK). We demonstrate that 1) EF24 induces death of lung, breast, ovarian, and cervical cancer cells, with a potency about 10 times higher than that of curcumin; 2) EF24 rapidly blocks the nuclear translocation of NF-B, with an IC₅₀ value of 1.3 µM compared with curcumin, with an IC₅₀ value of 13 µM; 3) EF24 effectively inhibits tumor necrosis factor (TNF)--induced IB phosphorylation and degradation, suggesting a role of this compound in targeting IKK; and 4) EF24 indeed directly inhibits the catalytic activity of IKK in an in vitro-reconstituted system. Our study identifies IKK as an effective target for EF24 and provides a molecular explanation for a superior activity of EF24 over curcumin. The effective inhibition of TNF--induced NF-B signaling by EF24 extends the therapeutic application of EF24 to other NF-B-dependent diseases, including inflammatory diseases such as rheumatoid arthritis.

[ARTICLES] Computational Discovery of Novel Low Micromolar Human Pregnane X Receptor Antagonists

2008-08-21

Very few antagonists have been identified for the human pregnane X receptor (PXR). These molecules may be of use for modulating the effects of therapeutic drugs, which are potent agonists for this receptor (e.g., some anticancer compounds and macrolide antibiotics), with subsequent effects on transcriptional regulation of xenobiotic metabolism and transporter genes. A recent novel pharmacophore for PXR antagonists was developed using three azoles and consisted of two hydrogen bond acceptor regions and two hydrophobic features. This pharmacophore also suggested an overall small binding site that was identified on the outer surface of the receptor at the AF-2 site and validated by docking studies. Using computational approaches to search libraries of known drugs or commercially available molecules is preferred over random screening. We have now described several new smaller antagonists of PXR discovered with the antagonist pharmacophore with in vitro activity in the low micromolar range [S-p-tolyl 3',5-dimethyl-3,5'-biisoxazole-4'-carbothioate (SPB03255) (IC₅₀, 6.3 µM) and 4-(3-chlorophenyl)-5-(2,4-dichlorobenzylthio)-4H-1,2,4-triazol-3-ol (SPB00574) (IC₅₀, 24.8 µM)]. We have also used our computational pharmacophore and docking tools to suggest that most of the known PXR antagonists, such as coumestrol and sulforaphane, could also interact on the outer surface of PXR at the AF-2 domain. The involvement of this domain was also suggested by further site-directed mutagenesis work. We have additionally described an FDA approved prodrug, leflunomide (IC₅₀, 6.8 µM), that seems to be a PXR antagonist in vitro. These observations are important for predicting whether further molecules may interact with PXR as antagonists in vivo with potential therapeutic applications.

[ARTICLES] Caffeine Stimulates Cytochrome Oxidase Expression and Activity in the Striatum in a Sexually Dimorphic Manner

Jones, F. S., Jing, J., Stonehouse, A. H., Stevens, A., Edelman, G. M. — 2008-08-21

Epidemiological studies indicate that caffeine consumption reduces the risk of Parkinson's disease (PD) in men, and antagonists of the adenosine 2A receptor ameliorate the motor symptoms of PD. These findings motivated us to identify proteins whose expression is regulated by caffeine in a sexually dimorphic manner. Using mass spectroscopy, we found that Cox7c, a nuclear-encoded subunit of the mitochondrial enzyme cytochrome oxidase, is up-regulated in the striatum of male but not female mice after receiving a single dose of caffeine. The expression of two other Cox subunits, Cox1 and Cox4, was also stimulated by caffeine in a male-specific fashion. This up-regulation of Cox subunits by caffeine was accompanied by an increase in Cox enzyme activity in the male striatum. Caffeine-induced stimulation of Cox expression and activity were reproduced using the adenosine 2A receptor (A2AR)-specific antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261), and coadministration of the A2AR-specific agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680) counteracted the elevation of Cox expression and activity by caffeine. Caffeine also increased Cox activity in PC-12 cells. In contrast, small interfering RNA (siRNA) knockdown of Cox7c expression in PC-12 cells blunted Cox activity, and this was counteracted by caffeine treatment. Caffeine was also found to increase Cox7c mRNA expression in the striatum and in PC-12 cells. This occurred at the level of transcription and was mediated by a segment of the Cox7c promoter. Overall, these findings indicate that cytochrome oxidase is a metabolic target of caffeine and that stimulation of Cox activity by caffeine via blockade of A2AR signaling may be an important mechanism underlying the therapeutic benefits of caffeine in PD.

[ARTICLES] Activation of the A3 Adenosine Receptor Suppresses Superoxide Production and Chemotaxis of Mouse Bone Marrow Neutrophils

van der Hoeven, D., Wan, T. C., Auchampach, J. A. — 2008-08-21

Adenosine is formed in injured/ischemic tissues, where it suppresses the actions of essentially all cells of the immune system. Most of the anti-inflammatory actions of adenosine have been attributed to signaling through the G_s protein-coupled A_2A adenosine receptor (AR). Here, we report that the A₃AR is highly expressed in murine neutrophils isolated from bone marrow. Selective activation of the A₃AR with (2S,3S,4R,5R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylic acid methylamide (CP-532,903) potently inhibited mouse bone marrow neutrophil superoxide generation and chemotaxis induced by various activating agents. The selectivity of CP-532,903 was confirmed in assays using neutrophils obtained from A_2AAR and A₃AR gene "knockout" mice. In a model of thioglycollate-induced inflammation, treating mice with CP-532,903 inhibited recruitment of leukocytes into the peritoneum by specifically activating the A₃AR. Collectively, our findings support the theory that the A₃AR contributes to the anti-inflammatory actions of adenosine on neutrophils and provide a potential mechanistic explanation for the efficacy of A₃AR agonists in animal models of inflammation (i.e., inhibition of neutrophil-mediated tissue injury).

[ARTICLES] Elevated Glutathione Levels Confer Cellular Sensitization to Cisplatin Toxicity by Up-Regulation of Copper Transporter hCtr1

2008-08-21

Previous studies have demonstrated that treating cultured cells with cisplatin (CDDP) up-regulated the expression of glutathione (GSH) and its de novo rate-limiting enzyme glutamate-cysteine ligase (GCL), which consists of a catalytic (GCLC) and a modifier (GCLM) subunit. It has also been shown that many CDDP-resistant cell lines exhibit high levels of GCLC/GCLM and GSH. Because the GSH system is the major intracellular regulator of redox conditions that serve as an important detoxification cytoprotector, these results have been taken into consideration that elevated levels of GCL/GSH are responsible for the CDDP resistance. In contrast to this context, we demonstrated here that overexpression of GSH by transfection with an expression plasmid containing the GCLC cDNA conferred sensitization to CDDP through up-regulation of human copper transporter (hCtr) 1, which is also a transporter for CDDP. Depleting GSH levels in these transfected cells reversed CDDP sensitivity with concomitant reduction of hCtr1 expression. Although rates of copper transport were also up-regulated in the transfected cells, these cells exhibited biochemical signature of copper deficiency, suggesting that GSH functions as an intracellular copper-chelator and that overexpression of GSH can alter copper metabolism. More importantly, our results reveal a new role of GSH in the regulation of CDDP sensitivity. Overproduction of GSH depletes the bioavailable copper pool, leading to up-regulation of hCtr1 and sensitization of CDDP transport and cell killing. These findings also have important implications in that modulation of the intracellular copper pool may be a novel strategy for improving chemotherapeutic efficacy of platinum-based antitumor agents.

[ARTICLES] Transcription Factor Sp1 Plays an Important Role in the Regulation of Copper Homeostasis in Mammalian Cells

2008-08-21

Copper is an essential metal nutrient, yet copper overload is toxic. Here, we report that human copper transporter (hCtr) 1 plays an important role in the maintenance of copper homeostasis by demonstrating that expression of hCtr1 mRNA was up-regulated under copper-depleted conditions and down-regulated under copper-replete conditions. Overexpression of full-length hCtr1 by transfection with a recombinant hCtr1 cDNA clone reduced endogenous hCtr1 mRNA levels, whereas overexpression of N terminus-deleted hCtr1 did not change endogenous hCtr1 mRNA levels, suggesting that increased functional hCtr1 transporter, which leads to increased intracellular copper content, down-regulates the endogenous hCtr1 mRNA. A luciferase assay using reporter constructs containing the hCtr1 promoter sequences revealed that three Sp1 binding sites are involved in the basal and copper concentration-dependent regulation of hCtr1 expression. Modulation of Sp1 levels affected the expression of hCtr1. We further demonstrated that the zinc-finger domain of Sp1 functions as a sensor of copper that regulates hCtr1 up and down in response to copper concentration variations. Our results demonstrate that mammalian copper homeostasis is maintained at the hCtr1 mRNA level, which is regulated by the Sp1 transcription factor.

[ARTICLES] Effect of Pregnancy on Cytochrome P450 3a and P-Glycoprotein Expression and Activity in the Mouse: Mechanisms, Tissue Specificity, and Time Course

Zhang, H., Wu, X., Wang, H., Mikheev, A. M., Mao, Q., Unadkat, J. D. — 2008-08-21

The plasma concentrations of orally administered anti-human immunodeficiency virus protease inhibitors are significantly reduced during human and mouse pregnancy. We have shown that in the mouse, at gestational day 19, this reduction is due to increased hepatic cytochrome P450 3a (Cyp3a) protein expression and activity. In the current study, we investigated the mechanisms by which Cyp3a activity is increased by pregnancy and the time course of change in expression of Cyp3a and P-glycoprotein (P-gp) in various tissues. We found that hepatic transcripts of Cyp3a16, Cyp3a41, and Cyp3a44 were significantly increased during pregnancy, whereas those of Cyp3a11 and Cyp3a25 were significantly decreased. This resulted in a net increase in Cyp3a protein expression and activity in the liver during pregnancy. The increase in Cyp3a41 and Cyp3a44 transcripts was positively correlated (p < 0.05) with hepatocyte nuclear factor 6 and estrogen receptor- transcripts. The pregnancy-related factors that transcriptionally activated mouse Cyp3a isoforms also activated the human CYP3A4 promoter in pregnant CYP3A4-promoter-luciferase transgenic (CYP3A4-tg) mice. In contrast, intestinal Cyp3a protein expression was not significantly affected by pregnancy. No change in P-gp protein expression was observed in the liver or kidney during pregnancy, although a significant decrease was observed in the placenta. Because hepatic CYP3A activity also seems to be induced during human pregnancy, the mouse (including CYP3A4-tg mouse) seems to be an excellent animal model to determine the molecular mechanisms for such an induction.

[ARTICLES] Overcoming S-Phase Checkpoint-Mediated Resistance: Sequence-Dependent Synergy of Gemcitabine and 7-Ethyl-10-hydroxycamptothecin (SN-38) in Human Carcinoma Cell Lines

2008-08-21

Although agents that inhibit DNA synthesis are widely used in the treatment of cancer, the optimal method for combining such agents and the mechanism of their synergy is poorly understood. The present study examined the effects of combining gemcitabine (2',2'-difluoro 2'-deoxycytidine) and 7-ethyl-10-hydroxycamptothecin (SN-38; the active metabolite of irinotecan), two S-phaseselective agents that individually have broad antitumor activity, in human cancer cells in vitro. Colony-forming assays revealed that simultaneous treatment of Ovcar-5 ovarian cancer cells or BxPC-3 pancreatic cancer cells with gemcitabine and SN-38 resulted in antagonistic effects. In contrast, sequential treatment with these two agents in either order resulted in synergistic anti-proliferative effects, although the mechanism of synergy varied with the sequence. In particular, SN-38 arrested cells in S phase, enhanced the accumulation of gemcitabine metabolites, and diminished checkpoint kinase 1, thereby sensitizing cells in the SN-38 -> gemcitabine sequence. Gemcitabine treatment followed by removal allowed prolonged progression through S phase, contributing to synergy of the gemcitabine -> SN-38 sequence. These results collectively suggest that S-phase-selective agents might exhibit more cytotoxicity when administered sequentially rather than simultaneously.

[ARTICLES] Sulfonylurea Receptor-Dependent and -Independent Pathways Mediate Vasodilation Induced by ATP-Sensitive K+ Channel Openers

Adebiyi, A., McNally, E. M., Jaggar, J. H. — 2008-08-21

ATP-sensitive K⁺ (K_ATP) channel openers are vasodilators that activate both plasma membrane and mitochondrial K_ATP channels. Here, we investigated the molecular mechanisms by which diazoxide and pinacidil induce vasodilation by studying diameter regulation of wild-type [SUR2(+/+)] and sulfonylurea receptor (SUR) 2-deficient [SUR2(-/-)] mouse myogenic mesenteric arteries. Ryanodine (10 µM), a ryanodine-sensitive Ca²⁺ release (RyR) channel blocker; iberiotoxin (100 nM), a large-conductance Ca²⁺-activated K⁺ (K_Ca) channel blocker; 4-aminopyridine (4-AP; 1 mM), a voltage-gated K⁺ (K_V) channel blocker; manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; 100 µM), an antioxidant; and a combination of ryanodine and 4-AP reduced diazoxide (100 µM)-induced dilation in pressurized (60 mm Hg) SUR2(+/+) arteries by 45 to 77%. In contrast, these inhibitors did not alter pinacidil (5 µM)-induced dilation in SUR2(+/+) arteries. Reverse transcription-polymerase chain reaction indicated that SUR2B was the only SUR isoform expressed in SUR2(+/+) mesenteric artery smooth muscle cells, whereas SURs were absent in SUR2(-/-) cells. In SUR2(-/-) arteries, pinacidil-induced vasodilation was ~10% of that in SUR2(+/+) arteries, whereas diazoxide-induced vasodilation was similar in SUR2(+/+) and SUR2(-/-) arteries. Atpenin (1 µM), a selective electron transport chain (ETC) complex II inhibitor, dilated arteries similarly to diazoxide, and this effect was attenuated by MnTMPyP and ryanodine + 4-AP. Atpenin also attenuated diazoxide-, but not pinacidil-induced vasodilation. In summary, data indicate that pinacidil-induced vasodilation requires SUR2B, whereas diazoxide-induced vasodilation does not require SURs. Rather, diazoxide-induced vasodilation involves ETC_II inhibition; a smooth muscle cell-reactive oxygen species elevation; and RyR, K_Ca, and K_V channel activation. These data indicate that K_ATP channel openers regulate arterial diameter via SUR-dependent and -independent pathways.

[ARTICLES] Novel Polymorphic Human UDP-glucuronosyltransferase 2A3: Cloning, Functional Characterization of Enzyme Variants, Comparative Tissue Expression, and Gene Induction

Court, M. H., Hazarika, S., Krishnaswamy, S., Finel, M., Williams, J. A. — 2008-08-21

UDP-glucuronosyltransferases (UGTs) are critical to the detoxification of numerous drugs, environmental pollutants, and endogenous molecules. However, as yet not all of the human UGTs have been cloned and characterized. cDNA clones from the UGT2A3 gene (located on chromosome 4q13) were isolated using pooled human liver RNA. Approximately 10% of clones contained a c.1489A>G nucleotide substitution, yielding proteins with a residue 497 alanine (UGT2A3.2) instead of a threonine (UGT2A3.1). The allele frequency of this polymorphism (rs13128286) was 0.13 in a European-American population as determined by direct DNA sequencing. Of 81 structurally diverse glucuronidation substrates tested, UGT2A3 expressed by a baculovirus system selectively glucuronidated bile acids, particularly hyodeoxycholic acid at the 6-hydroxy position. Apparent K_m values of UGT2A3.1 and UGT2A3.2 for hyodeoxycholic acid 6-glucuronidation were 69 ± 7 and 44 ± 12 µM, respectively. Of 29 different extrahepatic tissues evaluated by real-time polymerase chain reaction, UGT2A3 mRNA was most highly expressed in small intestine (160% of liver), colon (78% of liver), and adipose tissue (91% of liver). An in silico scan of the proximal UGT2A3 promoter/5'-regulatory region identified transcription factor consensus elements consistent with tissue-selective expression in liver (HNF1) and intestine (CXD2), as well as induction by rifampicin (pregnane X receptor). In LS180 human intestinal cells, rifampicin increased UGT2A3 mRNA by more than 4.5-fold compared with vehicle, whereas levels were not significantly affected by the arylhydrocarbon receptor ligand β-naphthoflavone. This is the first report establishing UGT2A3 as a functional enzyme, and it represents significant progress toward the goal of having a complete set of recombinant human UGTs for comparative functional analyses.

[ARTICLES] Aging-Resistant Organophosphate Bioscavenger Based on Polyethylene Glycol-Conjugated F338A Human Acetylcholinesterase

2008-08-21

The high reactivity of cholinesterases (ChEs) toward organophosphorus (OP) compounds has led to propose recombinant ChEs as bioscavengers of nerve agents. The bioscavenging potential of recombinant ChEs can be enhanced by conjugation of polyethylene glycol (PEG) moieties, to extend their circulatory residence. However, the ability of exogenously administered ChEs to confer long-term protection against repeated exposures to nerve agents is still limited due to the aging process, whereby organophosphate-ChE adducts undergo irreversible dealkylation, which precludes oxime-mediated reactivation of the enzyme. To generate an optimal acetylcholinesterase (AChE)-based OP bioscavenger, the F338A mutation, known to decelerate the rate of aging of AChE-OP conjugates, was incorporated into polyethylene glycol-conjugated (PEGylated) human AChE. The PEGylated F338A-AChE displayed unaltered rates of hydrolysis, inhibition, phosphylation, and reactivation and could effectively protect mice against exposure to soman (pinacolylmethyl phosphonofluoridate), sarin (O-isopropyl methylphosphonofluoridate), or O-ethyl-S-(2-isopropylaminoethyl) methylphosphonothioate (VX). Unlike PEGylated wild-type (WT)-AChE, the PEGylated F338A-AChE exhibits significantly reduced aging rates after soman inhibition and can be efficiently reactivated by the 1-[[[4(aminocarbonyl)-pyridinio]methoxy]methyl]-2(hydroxyimino)methyl]pyridinium dichloride (HI-6) oxime, both in vitro and in vivo. Accordingly, oxime administration to PEG-F338A-AChE-pretreated mice enabled them to withstand repeated soman exposure (5.4 and 4 LD₅₀/dose), whereas same regime treatment of non-PEGylated F338A-AChE- or PEGylated WT-AChE-pretreated mice failed to protect against the second challenge, due to rapid clearance or irreversible aging of the latter enzymes. Thus, judicious incorporation of selected mutations into the AChE mold in conjunction with its chemical modification provides means to engineer an optimal ChE-based OP bioscavenger in terms of circulatory longevity, resistance to aging, and reduced doses required for protection, even against repeated exposures to nerve agents, such as soman.

[ARTICLES] Activation and Block of the Adult Muscle-Type Nicotinic Receptor by Physostigmine: Single-Channel Studies

Militante, J., Ma, B.-W., Akk, G., Steinbach, J. H. — 2008-08-21

The plant-derived acetylcholinesterase inhibitor physostigmine has previously been shown to act on the nicotinic acetylcholine receptor (nAChR) causing either direct activation or potentiation of currents elicited by low concentrations of nicotinic agonists, or, at higher concentrations, channel block. We examined mouse adult-type muscle nAChR activation by physostigmine and found that channel activation by physostigmine exhibits many characteristics common with channel activity elicited by nicotinic agonists. Single-channel conductance was indistinguishable, and mutants known to slow channel closing in the presence of nicotinic agonists had a similar effect in the presence of physostigmine. However, physostigmine is a very inefficacious agonist. The presence of physostigmine did not alter the effective opening rate for a subsaturating dosage of carbachol, suggesting that physostigmine does not interact with the nicotinic agonist binding site. Mutations to a residue (Lys125) previously identified as part of the putative binding site for physostigmine reduced the duration of openings elicited by physostigmine, but the effects were generally small and, in most cases, nonsignificant. At higher concentrations, physostigmine blocked channel activity. Block manifested as a reduction in the mean open time and the emergence of a closed state, with a mean duration of 3 to 7 ms. The properties of block were consistent with two equivalent blocking sites per receptor with microscopic binding and unbinding rate constants for physostigmine of 20 µM^-1 s^-1 and 450 s^-1 (K_D = 23 µM). These observations indicate that physostigmine is able to activate muscle nAChR by interacting with a site other than the nicotinic ligand binding site.

[ARTICLES] Knockout Mice Reveal a Role for P2Y6 Receptor in Macrophages, Endothelial Cells, and Vascular Smooth Muscle Cells

2008-08-21

P2Y receptors are G-protein-coupled receptors activated by extracellular nucleotides. The P2Y₆ receptor is selectively activated by UDP, and its transcript has been detected in numerous organs, including the spleen, thymus, intestine, blood leukocytes, and aorta. To investigate the biological functions of this receptor, we generated P2Y₆-null mice by gene targeting. The P2Y₆ knockout (KO) mice are viable and are not distinguishable from the wild-type (WT) mice in terms of growth or fertility. In thioglycollate-elicited macrophages, the production of inositol phosphate in response to UDP stimulation was lost, indicating that P2Y₆ is the unique UDP-responsive receptor expressed by mouse macrophages. Furthermore, the amount of interleukin-6 and macrophage-inflammatory protein-2, but not tumor necrosis factor-, released in response to lipopolysaccharide stimulation was significantly enhanced in the presence of UDP, and this effect was lost in the P2Y₆ KO macrophages. The endothelium-dependent relaxation of the aorta by UDP was abolished in KO P2Y₆ mice. The contractile effect of UDP on the aorta, observed when endothelial nitric-oxide synthase is blocked, was also abolished in P2Y₆-null mice. In conclusion, we generated P2Y₆-deficient mice and have shown that these mice have a defective response to UDP in macrophages, endothelial cells, and vascular smooth muscle cells. These observations might be relevant to several physiopathological conditions such as atherosclerosis or hypertension.

[ARTICLES] Direct Modulation of P2X1 Receptor-Channels by the Lipid Phosphatidylinositol 4,5-Bisphosphate

2008-08-21

The P2X₁ receptor-channels activated by extracellular ATP contribute to the neurogenic component of smooth muscle contraction in vascular beds and genitourinary tracts of rodents and humans. In the present study, we investigated the interactions of plasma membrane phosphoinositides with P2X₁ ATP receptors and their physiological consequences. In an isolated rat mesenteric artery preparation, we observed a strong inhibition of P2X₁-mediated constrictive responses by depletion of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] with the phosphatidylinositol 4-kinase inhibitor wortmannin. Using the Xenopus laevis oocyte expression system, we provided electrophysiological evidence that lowering PI(4,5)P₂ levels with wortmannin significantly decreases P2X₁ current amplitude and recovery. Previously reported modulation of recovery of desensitized P2X₁ currents by phospholipase C-coupled 5-hydroxytryptamine_2A metabotropic receptors was also found to be wortmannin-sensitive. Treatment with wortmannin alters the kinetics of P2X₁ activation and inactivation without changing its sensitivity to ATP. The functional impact of wortmannin on P2X₁ currents could be reversed by addition of intracellular PI(4,5)P₂, but not phosphatidylinositol 3,4,5-trisphosphate, and direct application of PI(4,5)P₂ to excised inside-out macropatches rescued P2X₁ currents from rundown. We showed that the proximal region of the intracellular C terminus of P2X₁ subunit directly binds to PI(4,5)P₂ and other anionic phospholipids, and we identified the basic residue Lys₃₆₄ as a critical determinant for phospholipid binding and sensitivity to wortmannin. Overall, these results indicate that PI(4,5)P₂ plays a key role in the expression of full native and heterologous P2X₁ function by regulating the amplitude, recovery, and kinetics of ionotropic ATP responses through direct receptor-lipid interactions.

[ARTICLES] Erlotinib Induces Mitochondrial-Mediated Apoptosis in Human H3255 Non-Small-Cell Lung Cancer Cells with Epidermal Growth Factor ReceptorL858R Mutation through Mitochondrial Oxidative Phosphorylation-Dependent Activation of BAX and BAK

Ling, Y.-H., Lin, R., Perez-Soler, R. — 2008-08-21

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib shows potent antitumor activity in some non-small-cell lung cancer (NSCLC) cell lines and is approved by the Food and Drug Administration as second and third line treatment for NSCLC. However, the molecular mechanisms by which erlotinib induces apoptosis remain to be elucidated. Here, we investigated the effect of erlotinib on apoptotic signal pathways in H3255 cells with the EGFR^L858R mutation. Erlotinib induces apoptosis associated with the activation of caspases in a dose- and time-dependent manner. Erlotinib did not alter the expression of apoptotic receptors FAS and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), although it induced caspase-8 activation and BID cleavage. In addition, cell death caused by erlotinib was not prevented by coincubation with FAS and TRAIL antagonists, ZB-4 monoclonal antibody and TRAIL/Fc recombinant, suggesting that erlotinib-induced apoptosis is not associated with receptor-mediated pathways. Erlotinib induces loss of mitochondrial membrane potential and release of cytochrome c and second mitochondria-derived activator of caspases/direct IAP binding protein with low pI from mitochondria. Furthermore, erlotinib causes BAX translocation to mitochondria, BAX and BAK conformational changes, and oligomerization. Erlotinib did not induce reactive oxygen species generation, and cotreatment with antioxidants did not alter erlotinib-induced activation of BAX and BAK and apoptosis. However, cotreatment with inhibitors of mitochondrial oxidative phosphorylation significantly blocked erlotinib-induced activation of BAX and BAK and cell death. Benzyloxycarbiny-VAD-fluoromethyl ketone had no effect on erlotinib-induced BAX and BAK activation but effectively prevented apoptosis. Overexpression of BCL-2 caused a significant attenuation of erlotinib-induced cell death, but no effect on BAX and BAK activation. Down-regulation of BAX and BAK gene expression with small interfering RNA led to an effective reduction of erlotinib-induced apoptosis. Our data indicate that activation of BAX and BAK plays a critical role in the initiation of erlotinib-induced apoptotic cascades.

[ARTICLES] Lapatinib Resistance in HCT116 Cells Is Mediated by Elevated MCL-1 Expression and Decreased BAK Activation and Not by ERBB Receptor Kinase Mutation

2008-08-21

We have defined some of the mechanisms by which the kinase inhibitor lapatinib kills HCT116 cells. Lapatinib inhibited radiation-induced activation of ERBB1/2, extracellular signal-regulated kinases 1/2, and AKT, and radiosensitized HCT116 cells. Prolonged incubation of HCT116 cells with lapatinib caused cell killing followed by outgrowth of lapatinib-adapted cells. Adapted cells were resistant to serum starvation-induced cell killing and were cross-resistant to multiple therapeutic drugs. Lapatinib was competent to inhibit basal and epidermal growth factor (EGF)-stimulated ERBB1 phosphorylation in adapted cells. Coexpression of dominant-negative ERBB1 and dominant-negative ERBB2 inhibited basal and EGF-stimulated ERBB1 and ERBB2 phosphorylation in parental and adapted cells. However, in neither parental nor adapted cells did expression of dominant-negative ERBB1 and dominant-negative ERBB2 recapitulate the cell death-promoting effects of lapatinib. Adapted cells had increased expression of MCL-1, decreased expression of BAX, and decreased activation of BAX and BAK. Overexpression of BCL-XL protected parental cells from lapatinib toxicity. Knockdown of MCL-1 expression enhanced lapatinib toxicity in adapted cells that was reverted by knockdown of BAK expression. Inhibition of caspase function modestly reduced lapatinib toxicity in parental cells, whereas knockdown of apoptosis-inducing factor expression suppressed lapatinib toxicity. Thus, in HCT116 cells, lapatinib adaptation can be mediated by altered expression of pro- and antiapoptotic proteins that maintain mitochondrial function.

[ARTICLES] Elevated GSH Level Increases Cadmium Resistance through Down-Regulation of Sp1-Dependent Expression of the Cadmium Transporter ZIP8

Aiba, I., Hossain, A., Kuo, M. T. — 2008-08-21

Cadmium is a nonessential toxic metal in mammals. Its toxicity is mainly caused by interactions with cellular proteins that result in protein dysfunction and then disturb normal cellular functions. Glutathione (GSH) has been reported to play a role in cadmium resistance by serving as a cofactor for multidrug resistance protein 1/GS-X pump-mediated cadmium elimination. To further investigate the role of GSH in cadmium toxicity, we carried out a comparative study using small-cell lung cancer-derived cell lines, SR3A, and those that were stably transfected with glutamate cysteine ligase catalytic subunit (GCLC), a rate-limiting enzyme in GSH biosynthesis. These GCLC stably transfected cell lines produced higher levels of GSH and were more resistant to cadmium toxicity than the parental cell line was. The rates of cadmium uptake were reduced in these GCLC-transfected cell lines, which were associated with down-regulation of the cadmium transporter ZIP8/SLC39A8. Further analyses demonstrated that Sp1 binding site at the proximal promoter region of ZIP8 was sensitive to the GSH level and that the expression level of transcription factor Sp1 was reduced by increased GSH levels. We also demonstrated that low concentrations of cadmium exposure down-regulated ZIP8 expression with concomitant reduction of Sp1 expression. Taken together, these results demonstrate the importance of Sp1 in the regulation of ZIP8 expression. More important, our results reveal a new mechanism by which elevated GSH levels confer cadmium resistance by down-regulation of ZIP8 expression through the suppression of Sp1.

[ARTICLES] Binding of Orthosteric Ligands to the Allosteric Site of the M2 Muscarinic Cholinergic Receptor

Redka, D. S., Pisterzi, L. F., Wells, J. W. — 2008-08-21

The M₂ muscarinic receptor has two topographically distinct sites: the orthosteric site and an allosteric site recognized by compounds such as gallamine. It also can exhibit cooperative effects in the binding of orthosteric ligands, presumably to the orthosteric sites within an oligomer. Such effects would be difficult to interpret, however, if those ligands also bound to the allosteric site. Monomers of the hemagglutinin (HA)- and FLAG-tagged human M₂ receptor therefore have been purified from coinfected Sf9 cells and examined for any effect of the antagonist N-methyl scopolamine or the agonist oxotremorine-M on the rate at which N-[³H]methyl scopolamine dissociates from the orthosteric site (k_obsd). The predominantly monomeric status was confirmed by coimmunoprecipitation and by cross-linking with bis(sulfosuccinimidyl)suberate. Both N-methyl scopolamine and oxotremorine-M acted in a cooperative manner to decrease k_obsd by 4.5- and 9.1-fold, respectively; the corresponding estimates of affinity (log K_L) are -2.55 ± 0.13 and -2.29 ± 0.14. Gallamine and the allosteric ligand obidoxime decreased k_obsd by more than 100-fold (log K_L = -4.12 ± 0.04) and by only 1.1-fold (log K_L = -1.73 ± 0.91), respectively. Obidoxime reversed the effect of N-methyl scopolamine, oxotremorine-M, and gallamine in a manner that could be described by a model in which all four ligands compete for a common allosteric site. Ligands generally assumed to be exclusively orthosteric therefore can act at the allosteric site of the M₂ receptor, albeit at comparatively high concentrations.

[ARTICLES] Long-Term Nicotine Treatment Differentially Regulates Striatal {alpha}6{alpha}4{beta}2* and {alpha}6(Non{alpha}4){beta}2* nAChR Expression and Function

Perez, X. A., Bordia, T., McIntosh, J. M., Grady, S. R., Quik, M. — 2008-08-21

Nicotine treatment has long been associated with alterations in 4β2^* nicotinic acetylcholine receptor (nAChR) expression that modify dopaminergic function. However, the influence of long-term nicotine treatment on the 6β2^* nAChR, a subtype specifically localized on dopaminergic neurons, is less clear. Here we used voltammetry, as well as receptor binding studies, to identify the effects of nicotine on striatal 6β2^* nAChR function and expression. Long-term nicotine treatment via drinking water enhanced nonburst and burst endogenous dopamine release from rat striatal slices. In control animals, 6β2^* nAChR blockade with -conotoxin MII (-CtxMII) decreased release with nonburst stimulation but not with burst firing. These data in control animals suggest that varying stimulus frequencies differentially regulate 6β2^* nAChR-evoked dopamine release. In contrast, in nicotine-treated rats, 6β2^* nAChR blockade elicited a similar pattern of dopamine release with nonburst and burst firing. To elucidate the 6β2^* nAChR subtypes altered with long-term nicotine treatment, we used the novel -CtxMII analog E11A in combination with 4 nAChR knockout mice. ¹²⁵I--CtxMII competition studies in striatum of knockout mice showed that nicotine treatment decreased the 64β2^* subtype but increased the 6(non4)β2^* nAChR population. These data indicate that 6β2^* nAChR-evoked dopamine release in nicotine-treated rats is mediated by the 6(non4)β2^* nAChR subtype and suggest that the 64β2^* nAChR and/or 4β2^* nAChR contribute to the differential effect of higher frequency stimulation on dopamine release under control conditions. Thus, 6β2^* nAChR subtypes may represent important targets for smoking cessation therapies and neurological disorders involving these receptors such as Parkinson's disease.

[ARTICLES] The Proton-Coupled Folate Transporter: Impact on Pemetrexed Transport and on Antifolates Activities Compared with the Reduced Folate Carrier

Zhao, R., Qiu, A., Tsai, E., Jansen, M., Akabas, M. H., Goldman, I. D. — 2008-08-21

The reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT) are ubiquitously expressed in normal and malignant mammalian tissues and in human solid tumor cell lines. This article addresses the extent to which PCFT contributes to transport of pemetrexed and to the activities of this and other antifolates relative to RFC at physiological pH. Either RFC or PCFT cDNA was stably transfected into a transporter-null HeLa cell variant to achieve activities similar to their endogenous function in wild-type HeLa cells. PCFT and RFC produced comparable increases in pemetrexed activity in growth medium with 5-formyltetrahydrofolate. However, PCFT had little or no effect on the activities of methotrexate, N-(5-[N-(3,4-dihydro-2-methyl-4-oxyquinazolin-6-ylmethyl)-N-methyl-amino]-2-thenoyl)-l-glutamic acid (raltitrexed, Tomudex; ZD1694), or N-(4-amino-4-deoxypteroyl)-N-hemiphthaloyl-l-ornithine (PT523) in comparison with RFC irrespective of the folate growth source. PCFT, expressed at high levels in Xenopus laevis oocytes and in transporter-competent HepG2 cells, exhibited a high affinity for pemetrexed, with an influx K_m value of 0.2 to 0.8 µM at pH 5.5. PCFT increased the growth inhibitory activity of pemetrexed, but not that of the other antifolates in HepG2 cells grown with 5-formyltetrahydrofolate at physiological pH. These findings illustrate the unique role that PCFT plays in the transport and pharmacological activity of pemetrexed. Because of the ubiquitous expression of PCFT in human tumors, and the ability of PCFT to sustain pemetrexed activity even in the absence of RFC, tumor cells are unlikely to become resistant to pemetrexed as a result of impaired transport because of the redundancy of these genetically distinct routes.

[ARTICLES] MLH1 Deficiency Enhances Radiosensitization with 5-Fluorodeoxyuridine by Increasing DNA Mismatches

Flanagan, S. A., Krokosky, C. M., Mannava, S., Nikiforov, M. A., Shewach, D. S. — 2008-08-21

The antitumor drug 5-fluoro-2'-deoxyuridine (FdUrd) also sensitizes tumor cells to ionizing radiation in vitro and in vivo. Although radiosensitization with FdUrd requires dTTP depletion and S-phase arrest, the exact mechanism by which these events produce radiosensitization remains unknown. We hypothesized that the depletion of dTTP produces DNA mismatches that, if not repaired before irradiation, would result in radiosensitization. We evaluated this hypothesis in mismatch repair (MMR)-deficient HCT116 0-1 cells that lack the expression of the required MMR protein MLH1 (inactive MLH1), and in MMR-proficient (wild-type MLH1) HCT116 1-2 cells. Although HCT116 0-1 cells were less sensitive to FdUrd (IC₅₀ = 3.5 µM) versus HCT116 1-2 cells (IC₅₀ = 0.75 µM), when irradiation followed FdUrd (IC₅₀) the MLH1-inactivated cells exhibited greater radiosensitization compared with MMR-wild-type cells [radiation enhancement ratio (RER) = 1.8 ± 0.28 versus 1.1 ± 0.1, respectively] and an increase (≥8-fold) in nucleotide misincorporations. In SW620 cells and HCT116 1-2 MLH1-wild-type cells, FdUrd (IC₅₀) did not produce radiosensitization nor did it increase the mutation frequency, but after short hairpin RNA-directed suppression of MLH1 this concentration produced excellent radiosensitization (RER = 1.6 ± 0.10 and 1.5 ± 0.06, respectively) and an increase in nucleotide misincorporations (8-fold and 6-fold, respectively). Incubation with higher concentrations of FdUrd (IC₉₀) after suppression of MLH1 produced a further increase in ionizing radiation sensitivity in both SW620 and HCT116 1-2 cells (RER = 1.8 ± 0.03 and 1.7 ± 0.13, respectively) and nucleotide misincorporations (>10-fold in both cell lines). These results demonstrate an important role for MLH1 and implicate mismatches in radiosensitization by FdUrd.

[ARTICLES] Combinatorial Antileukemic Disruption of Oxidative Homeostasis and Mitochondrial Stability by the Redox Reactive Thalidomide 2-(2,4-Difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione (CPS49) and Flavopiridol

2008-08-21

2-(2,4-Difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione (CPS49) is a member of a recently identified class of redox-reactive thalidomide analogs that show selective killing of leukemic cells by increasing intracellular reactive oxygen species (ROS) and targeting multiple transcriptional pathways. Flavopiridol is a semisynthetic flavonoid that inhibits cyclin-dependent kinases and also shows selective lethality against leukemic cells. The purpose of this study is to explore the efficacy and mechanism of action of the combinatorial use of the redox-reactive thalidomide CPS49 and the cyclin-dependent kinase inhibitor flavopiridol as a selective antileukemic therapeutic strategy. In combination, CPS49 and flavopiridol were found to induce selective cytotoxicity associated with mitochondrial dysfunction and elevations of ROS in leukemic cells ranging from additive to synergistic activity at low micromolar concentrations. Highest synergy was observed at the level of ROS generation with a strong correlation between cell-specific cytotoxicity and reciprocal coupling of drug-induced ROS elevation with glutathione depletion. Examination of the transcriptional targeting of CPS49 and flavopiridol combinations reveals that the drugs act in concert to initiate a cell specific transcriptional program that manipulates nuclear factor-B (NF-B), E2F-1, and p73 activity to promote enhanced mitochondrial instability by simultaneously elevating the expression of the proapoptotic factors BAX, BAD, p73, and PUMA while depressing expression of the antiapoptotic genes MCL1, XIAP, BCL-xL, SURVIVIN, and MDM2. The coadministration of CPS49 and flavopiridol acts through coordinate targeting of transcriptional pathways that enforce selective mitochondrial dysfunction and ROS elevation and is therefore a promising new therapeutic combination that warrants further preclinical exploration.

[ARTICLES] Proapoptotic Activity and Chemosensitizing Effect of the Novel Akt Inhibitor (2S)-1-(1H-Indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan2-amine (A443654) in T-Cell Acute Lymphoblastic Leukemia

2008-08-21

Constitutively activated AKT kinase is a common feature of T-cell acute lymphoblastic leukemia (T-ALL). Here, we report that the novel AKT inhibitor (2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan2-amine (A443654) leads to rapid cell death of T-ALL lines and patient samples. Treatment of CEM, Jurkat, and MOLT-4 cells with nanomolar doses of the inhibitor led to AKT phosphorylation accompanied by dephosphorylation and activation of the downstream target, glycogen synthase kinase-3β. Effects were time- and dose-dependent, resulting in apoptotic cell death. Treatment of Jurkat cells with A443654 resulted in activation of caspase-2, -3, -6, -8, and -9. Apoptotic cell death was mostly dependent on caspase-2 activation, as demonstrated by preincubation with a selective pharmacological inhibitor. It is remarkable that A443654 was highly effective against the drug-resistant cell line CEM-VBL100, which expresses 170-kDa P-glycoprotein. Moreover, A443654 synergized with the DNA-damaging agent etoposide in both drug-sensitive and drug-resistant cell lines when coadministered [combination index (CI) = 0.39] or when pretreated with etoposide followed by A443654 (CI = 0.689). The efficacy of A443654 was confirmed using blasts from six patients with T-ALL, all of whom displayed low levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and constitutive phosphorylation of Akt on Ser473. At 1 µM, the inhibitor was able to induce apoptotic cell death of T-ALL blast cells, as indicated by flow cytometric analysis of samples immunostained for active (cleaved) caspase-3. Because activated AKT is seen in a large percentage of patients with T-ALL, A443654, either alone or in combination with existing drugs, may be a useful therapy for primary and drug-resistant T-ALL.

[ARTICLES] Local Not Systemic Modulation of Dendritic Cell S1P Receptors in Lung Blunts Virus-Specific Immune Responses to Influenza

2008-08-21

The mechanism by which locally delivered sphingosine analogs regulate host response to localized viral infection has never been addressed. In this report, we show that intratracheal delivery of the chiral sphingosine analog (R)-2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol (AAL-R) or its phosphate ester inhibits the T-cell response to influenza virus infection. In contrast, neither intraperitoneal delivery of AAL-R nor intratracheal instillation of the non-phosphorylatable stereoisomer AAL-S suppressed virus-specific T-cell response, indicating that in vivo phosphorylation of AAL-R and sphingosine 1-phosphate (S1P) receptor modulation in lungs is essential for immunomodulation. Intratracheal delivery of water-soluble S1P₁ receptor agonist at doses sufficient to induce systemic lymphopenia did not inhibit virus-specific T-cell response, indicating that S1P₁ is not involved in the immunosuppressive activities of AAL-R and that immunosuppression acts independently of naive lymphocyte recirculation. Accumulation of dendritic cells (DCs) in draining lymph nodes was inhibited by intratracheal but not intraperitoneal delivery of AAL-R. Direct modulation of DCs is demonstrated by the impaired ability of virus-infected bone marrow-derived DCs treated in vitro with AAL-R to trigger in vivo T-cell response after adoptive transfer to the airways. Thus, our results suggest that locally delivered sphingosine analogs induce immunosuppression by modulating S1P receptors other than S1P₁ or S1P₂ on dendritic cells in the lungs after influenza virus infection.

[ARTICLES] Alternative Splicing of the Ca2+ Channel {beta}4 Subunit Confers Specificity for Gabapentin Inhibition of Cav2.1 Trafficking

Mich, P. M., Horne, W. A. — 2008-08-21

Gabapentin is well established as an effective treatment for neuropathic pain; however, little is known about its mechanism of action. It binds with high affinity to Ca²⁺ channel 2 subunits that are expressed in dorsal root ganglia. Mutation of a single 2 amino acid, R217A, eliminates both gabapentin binding and analgesic efficacy. Gabapentin does not seem to have direct Ca²⁺ channel blocking properties but does affect overall levels of Ca²⁺ channel surface expression in some circumstances. In this report, we examined gabapentin effects on trafficking and voltage-dependent gating properties of recombinant Ca_v2.1 Ca²⁺ channel complexes transiently expressed in Xenopus laevis oocytes. We also determined electrophysiologically whether gabapentin causes displacement of β subunits from Ca_v2.1 complexes. Our principal findings are as follows: 1) gabapentin inhibits trafficking of recombinant Ca_v2.1 Ca²⁺ channels in X. laevis oocytes; 2) gabapentin inhibition occurs in the presence of the Ca²⁺ channel β4a subunit but not in the presence of β4b; 3) gabapentin does not affect Ca_v2.1 voltage-dependent gating parameters; 4) inhibition of Ca_v2.1trafficking is highly dependent on β-subunit concentration; and 5) gabapentin inhibition of Ca_v2.1 trafficking can be reversed by the 2 R217A mutation. Overall, our results suggest that gabapentin reduces the number of β4a-bound Ca_v2.1 complexes that are successfully trafficked to the plasma membrane. This mechanism may help to explain why gabapentin is both effective and selective in the treatment of neuropathic pain states that involve up-regulation of 2 subunits.

[ARTICLES] Nuclear Receptor Coactivator 6 Mediates the Synergistic Activation of Human Cytochrome P-450 2C9 by the Constitutive Androstane Receptor and Hepatic Nuclear Factor-4{alpha}

Surapureddi, S., Rana, R., Reddy, J. K., Goldstein, J. A. — 2008-08-21

Nuclear receptor coactivator 6 (NCOA6) also known as PRIP/RAP250/ASC-2 anchors a steady-state complex of cofactors and function as a transcriptional coactivator for certain nuclear receptors. This is the first study to identify NCOA6 as a hepatic nuclear factor 4 (HNF4)-interacting protein. CYP2C9 is an important enzyme that metabolizes both commonly used therapeutic drugs and important endogenous compounds. We have shown previously that constitutive androstane receptor (CAR) (a xenobiotic-sensing receptor) up-regulates the CYP2C9 promoter through binding to a distal site, whereas HNF4 transcriptionally up-regulates CYP2C9 via proximal sites. We demonstrate ligand-enhanced synergistic cross-talk between CAR and HNF4. We identify NCOA6 as crucial to the underlying mechanism of this cross-talk. NCOA6 was identified as an HNF4-interacting protein in this study using a yeast two-hybrid screen and GST pull-down assays. Furthermore, we identified NCOA6, CAR, and other coactivators as part of a mega complex of cofactors associated with HNF4 in HepG2 cells. Although the interaction of NCOA6 with CAR is specifically through the first LXXLL motif of NCOA6, both LXXLL motifs are involved in its interaction with HNF4. Silencing of NCOA6 abrogated the synergistic activation of the CYP2C9 promoter and the synergistic induction of the CYP2C9 gene by CAR-HNF4. Chromatin immunoprecipitation analysis revealed that NCOA6 can pull down both the proximal HNF4 and distal CAR binding sites of the CYP2C9 promoter and provides the basis for the recruitment of other cofactors. We conclude that the coactivator NCOA6 mediates the mechanism of the synergistic activation of the CYP2C9 gene by CAR and HNF4.

[ERRATUM] Correction to "Fluvastatin Synergistically Improves the Antiproliferative Effect of Everolimus on Rat Smooth Muscle Cells by Altering p27Kip1/Cyclin E Expression"

2008-08-21

[ERRATUM] Correction to "Slc39a14 Gene Encodes ZIP14, A Metal/Bicarbonate Symporter: Similarities to the ZIP8 Transporter"

2008-08-21