Molecular Pharmacology current issue

[ARTICLES] Amitriptyline Activates Cardiac Ryanodine Channels and Causes Spontaneous Sarcoplasmic Reticulum Calcium Release

12/19/2008 12:00:00 AM

Patients taking amitriptyline (AMT) have an increased risk of sudden cardiac death, yet the mechanism for AMT's proarrhythmic effects remains incompletely understood. Here, we hypothesize that AMT activates cardiac ryanodine channels (RyR2), causing premature Ca²⁺ release from the sarcoplasmic reticulum (SR), a mechanism identified by genetic studies as a cause of ventricular arrhythmias and sudden cardiac death. To test this hypothesis, we measured the effect of AMT on RyR2 channels from mice and sheep and on intact mouse cardiomyocytes loaded with the Ca²⁺ fluorescent indicator Fura-2 acetoxymethyl ester. AMT induced trains of long channel openings (bursts) with 60 to 90% of normal conductance in RyR2 channels incorporated in lipid bilayers. The [AMT], voltage, and open probability (P_o) dependencies of burst frequency and duration indicated that AMT binds primarily to open RyR2 channels. AMT also activated RyR2 channels isolated from transgenic mice lacking cardiac calsequestrin. Reducing RyR2 P_o by increasing cytoplasmic [Mg²⁺] significantly inhibited the AMT effect on RyR2 channels. Consistent with the single RyR2 channel data, AMT increased the rate of spontaneous Ca²⁺ releases and decreased the SR Ca²⁺ content in intact cardiomyocytes. Intracellular [AMT] were approximately 5-fold higher than extracellular [AMT], explaining AMT's higher potency in cardiomyocytes at clinically relevant concentrations (0.5-3 µM) compared with its effect in lipid bilayers (5-10 µM). Increasing extracellular [Mg²⁺] attenuated the effect of AMT in intact myocytes. We conclude that the heretofore unrecognized activation of RyR2 channels and increased SR Ca²⁺ leak may contribute to AMT's proarrhythmic and cardiotoxic effects, which may be counteracted by interventions that reduce RyR2 channel open probability.

[ARTICLES] Fibroblast Growth Factor (FGF) and FGF Receptor-Mediated Autocrine Signaling in Non-Small-Cell Lung Cancer Cells

12/19/2008 12:00:00 AM

Despite widespread expression of epidermal growth factor (EGF) receptors (EGFRs) and EGF family ligands in non-small-cell lung cancer (NSCLC), EGFR-specific tyrosine kinase inhibitors (TKIs) such as gefitinib exhibit limited activity in this cancer. We propose that autocrine growth signaling pathways distinct from EGFR are active in NSCLC cells. To this end, gene expression profiling revealed frequent coexpression of specific fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in NSCLC cell lines. It is noteworthy that FGF2 and FGF9 as well as FGFR1 IIIc and/or FGFR2 IIIc mRNA and protein are frequently coexpressed in NSCLC cell lines, especially those that are insensitive to gefitinib. Specific silencing of FGF2 reduced anchorage-independent growth of two independent NSCLC cell lines that secrete FGF2 and coexpress FGFR1 IIIc and/or FGFR2 IIIc. Moreover, a TKI [(±)-1-(anti-3-hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1H-pyrimido-[4,5-d]pyrimidin-2-one (RO4383596)] that targets FGFRs inhibited basal FRS2 and extracellular signal-regulated kinase phosphorylation, two measures of FGFR activity, as well as proliferation and anchorage-independent growth of NSCLC cell lines that coexpress FGF2 or FGF9 and FGFRs. By contrast, RO4383596 influenced neither signal transduction nor growth of NSCLC cell lines lacking FGF2, FGF9, FGFR1, or FGFR2 expression. Thus, FGF2, FGF9 and their respective high-affinity FGFRs comprise a growth factor autocrine loop that is active in a subset of gefitinib-insensitive NSCLC cell lines.

[ARTICLES] YC-1 Stimulates the Expression of Gaseous Monoxide-Generating Enzymes in Vascular Smooth Muscle Cells

12/19/2008 12:00:00 AM

The benzylindazole derivative 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) is an allosteric stimulator of soluble guanylate cyclase (sGC) that sensitizes the enzyme to the gaseous ligands carbon monoxide (CO) and nitric oxide (NO). In this study, we examined whether YC-1 also promotes the production of these gaseous monoxides by stimulating the expression of the inducible isoforms of heme oxygenase (HO-1) and NO synthase (iNOS) in vascular smooth muscle cells (SMCs). YC-1 increased HO-1 mRNA, protein, and promoter activity and potentiated cytokine-mediated expression of iNOS protein and NO synthesis by SMCs. The induction of HO-1 by YC-1 was unchanged by the sGC inhibitor, 1H-(1,2,4)oxadiazolo[4,3-]quinozalin-1-one (ODQ) or by the protein kinase G inhibitors (8R,9S,11S)-(-)-2-methyl-9-methoxyl-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cyclocta9(cde)trinen-1-one (KT 5823) and YGRKKRRQRRRPPLRKKKKKH-amide (DT-2) and was not duplicated by 8-bromo-cGMP or the NO-independent sGC stimulator 5-cyclopropyl-2[1-(2-fluorobenzyl)-1H-pyrazolo [3,4-b] pyridine-3-yl] pyrimidin-4-ylamine (BAY 41-2272). However, the YC-1-mediated induction of HO-1 was inhibited by the phosphatidylinositol-3-kinase (PI3K) inhibitors wortmannin and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002). In contrast, the enhancement of cytokine-stimulated iNOS expression and NO production by YC-1 was prevented by ODQ and the protein kinase A inhibitor (9S,10S, 12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9, 12-epoxy-1H-diindolo(1,2,3-fg:3',2',1'-kl)pyrrolo(3,4-i)(1,6)-benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) and was mimicked by 8-bromo-cGMP and BAY 41-2272. In conclusion, these studies demonstrate that YC-1 stimulates the expression of HO-1 and iNOS in vascular SMCs via the PI3K and sGC-cGMP-protein kinase A pathway, respectively. The ability of YC-1 to sensitize sGC to gaseous monoxides and simultaneously stimulate their production through the induction of HO-1 and iNOS provides a potent mechanism by which the cGMP-dependent and -independent biological actions of this agent are amplified.

[ARTICLES] Copper Regulation of Hypoxia-Inducible Factor-1 Activity

12/19/2008 12:00:00 AM

Previous studies have demonstrated that copper up-regulates hypoxia-inducible factor 1 (HIF-1). The present study was undertaken to test the hypothesis that copper is required for HIF-1 activation. Treatment of HepG2 cells with a copper chelator tetraethylenepentamine (TEPA) or short interfering RNA targeting copper chaperone for superoxide dismutase 1 (CCS) suppressed hypoxia-induced activation of HIF-1. Addition of excess copper relieved the suppression by TEPA, but not that by CCS gene silencing, indicating the requirement of copper for activation of HIF-1, which is CCS-dependent. Copper deprivation did not affect production or stability of HIF-1 but reduced HIF-1 binding to the hypoxia-responsive element (HRE) of target genes and to p300, a component of HIF-1 transcriptional complex. Copper probably inhibits the factor inhibiting HIF-1 to ensure the formation of HIF-1 transcriptional complex. This study thus defines that copper is required for HIF-1 activation through the regulation of HIF-1 binding to the HRE and the formation of the HIF-1 transcriptional complex.

[ARTICLES] Vanillin Inhibits Matrix Metalloproteinase-9 Expression through Down-Regulation of Nuclear Factor-{kappa}B Signaling Pathway in Human Hepatocellular Carcinoma Cells

12/19/2008 12:00:00 AM

Vanillin has been reported to exhibit anti-invasive and antimetastatic activities by suppressing the enzymatic activity of matrix metalloproteinase-9 (MMP-9). However, the underlying mechanism of anti-invasive activity remains unclear so far. Herein we demonstrate that vanillin reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 gelatinolytic activity and suppressed cell invasion through the down-regulation of MMP-9 gene transcription in HepG2 cells. Vanillin significantly reduced the 6.6-fold invasive capacity of HepG2 cells in noncytotoxic concentrations, and this anti-invasive effect was concentration-dependent in the Matrigel invasion assay. Moreover, vanillin significantly suppressed the TPA-induced enzymatic activity of MMP-9 and decreased the induced mRNA level of MMP-9. Analysis of the transcriptional regulation indicated that vanillin suppressed MMP-9 transcription by inhibiting nuclear factor-B (NF-B) activity. Western blot further confirmed that vanillin inhibited NF-B activity through the inhibition of IB- phosphorylation and degradation. In conclusion, vanillin might be a potent antiinvasive agent that suppresses the MMP-9 enzymatic activity via NF-B signaling pathway.

[ARTICLES] Stereochemistry of an Agonist Determines Coupling Preference of {beta}2-Adrenoceptor to Different G Proteins in Cardiomyocytes

12/19/2008 12:00:00 AM

A fundamental question regarding receptor-G protein interaction is whether different agonists can lead a receptor to different intracellular signaling pathways. Our previous studies have demonstrated that although most β₂-adrenoceptor agonists activate both G_s and G_i proteins, fenoterol, a full agonist of β₂-adrenoceptor, selectively activates G_s protein. Fenoterol contains two chiral centers and may exist as four stereoisomers. We have synthesized a series of stereoisomers of fenoterol and its derivatives and characterized their receptor binding and pharmacological properties. We tested the hypothesis that the stereochemistry of an agonist determines selectivity of receptor coupling to different G protein(s). We found that the R,R isomers of fenoterol and methoxyfenoterol exhibited more potent effects to increase cardiomyocyte contraction than their S,R isomers. It is noteworthy that although (R,R)-fenoterol and (R,R)-methoxyfenoterol preferentially activate G_s signaling, their S,R isomers were able to activate both G_s and G_i proteins as evidenced by the robust pertussis toxin sensitivities of their effects on cardiomyocyte contraction and on phosphorylation of extracellular signal-regulated kinase 1/2. The differential G protein selectivities of the fenoterol stereoisomers were further confirmed by photoaffinity labeling studies on G_s,G_i2, and G_i3 proteins. The inefficient G_i signaling with the R,R isomers is not caused by the inability of the R,R isomers to trigger the protein kinase A (PKA)-mediated phosphorylation of the β₂-adrenoceptor, because the R,R isomers also markedly increased phosphorylation of the receptor at serine 262 by PKA. We conclude that in addition to receptor subtype and phosphorylation status, the stereochemistry of a given agonist plays an important role in determining receptor-G protein selectivity and downstream signaling events.

[ARTICLES] Site Selectivity of Competitive Antagonists for the Mouse Adult Muscle Nicotinic Acetylcholine Receptor

12/19/2008 12:00:00 AM

The muscle-type nicotinic acetylcholine receptor has two nonidentical binding sites for ligands. The selectivity of acetylcholine and the competitive antagonists (+)-tubocurarine and metocurine for adult mouse receptors is known. Here, we examine the site selectivity for four other competitive antagonists: cisatracurium, pancuronium, vecuronium, and rocuronium. We rapidly applied acetylcholine to outside-out patches from transfected BOSC23 cells and measured macroscopic currents. We have reported the IC₅₀ of the antagonists individually in prior publications. Here, we determined inhibition by pairs of competitive antagonists. At least one antagonist was present at a concentration producing ≥67% receptor inhibition. Metocurine shifted the apparent IC₅₀ of (+)-tubocurarine in quantitative agreement with complete competitive antagonism. The same was observed for pancuronium competing with vecuronium. However, pancuronium and vecuronium each shifted the apparent IC₅₀ of (+)-tubocurarine less than expected for complete competition but more than expected for independent binding. The situation was similar for cisatracurium and (+)-tubocurarine or metocurine. Cisatracurium did not shift the apparent IC₅₀ of pancuronium or vecuronium, indicating independent binding of these two pairs. The data were fit to a two-site, two-antagonist model to determine the antagonist binding constants for each site, L and L. We found L/L = 0.22 (range, 0.14-0.34), 20 (9-29), 21 (4-36), and 1.5 (0.3-2.9) for cisatracurium, pancuronium, vecuronium, and rocuronium, respectively. The wide range of L/L for some antagonists may reflect experimental uncertainties in the low affinity site, relatively poor selectivity (rocuronium), or possibly that the binding of an antagonist at one site affects the affinity of the second site.

[ERRATUM] Correction to "The NR1 M3 Domain Mediates Allosteric Coupling in the N-Methyl-D-aspartate Receptor"

12/19/2008 12:00:00 AM

[ERRATUM] Correction to "Inhibitors of GlyT1 Affect Glycine Transport via Discrete Binding Sites"

12/19/2008 12:00:00 AM

[ERRATUM] Correction to Table of Contents (Volume 74, Number 6, December 2008)

12/19/2008 12:00:00 AM

[ARTICLES] Inhibition of Arachidonic Acid and Iron-Induced Mitochondrial Dysfunction and Apoptosis by Oltipraz and Novel 1,2-Dithiole-3-thione Congeners

12/19/2008 12:00:00 AM

4-Methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione (oltipraz), a prototype drug candidate containing a 1,2-dithiole-3-thione moiety, has been widely studied as a cancer chemopreventive agent. Oltipraz and other novel 1,2-dithiole-3-thione congeners have the capability to prevent insulin resistance via AMP-activated protein kinase (AMPK) activation. Arachidonic acid (AA, a proinflammatory fatty acid) exerts a deleterious effect on mitochondria and promotes reactive oxygen species (ROS) production. This study investigated whether AA alone or in combination with iron (catalyst of autooxidation) causes ROS-mediated mitochondrial impairment, and if so, whether oltipraz and synthetic 1,2-dithiole-3-thiones protect mitochondria and cells against excess ROS produced by AA + iron. Oltipraz treatment effectively inhibited mitochondrial permeability transition promoted by AA + iron in HepG2 cells, thereby protecting cells from ROS-induced apoptosis. Oltipraz was found to attenuate apoptosis induced by rotenone (complex I inhibitor), but not that by antimycin A (complex III inhibitor), suggesting that the inhibition of AA-induced apoptosis by oltipraz might be associated with the electron transport system. AMPK activation by oltipraz contributed to cell survival, which was supported by the reversal of oltipraz's restoration of mitochondrial membrane potential by concomitant treatment of compound C. By the same token, an AMPK activator inhibited AA + iron-induced mitochondrial permeability transition with an increase in cell viability. Moreover, new 1,2-dithiole-3-thiones with the capability of AMPK activation protected cells from mitochondrial permeability transition and ROS overproduction induced by AA + iron. Our results demonstrate that oltipraz and new 1,2-dithiole-3-thiones are capable of protecting cells from AA + iron-induced ROS production and mitochondrial dysfunction, which may be associated with AMPK activation.

[ARTICLES] Intracellular Activation and Deactivation of Tasidotin, an Analog of Dolastatin 15: Correlation with Cytotoxicity

12/19/2008 12:00:00 AM

Tasidotin, an oncolytic drug in phase II clinical trials, is a peptide analog of the antimitotic depsipeptide dolastatin 15. In tasidotin, the carboxyl-terminal ester group of dolastatin 15 has been replaced by a carboxy-terminal tert-butyl amide. As expected from studies with cemadotin, [³H]tasidotin, with the radiolabel in the second proline residue, was hydrolyzed intracellularly, with formation of N,N-dimethylvalyl-valyl-N-methylvalyl-prolyl-proline (P5), a pentapeptide also present in dolastatin 15 and cemadotin. P5 was more active as an inhibitor of tubulin polymerization and less active as a cytotoxic agent than tasidotin, cemadotin, and dolastatin 15. [³H]P5 was not the end product of tasidotin metabolism. Large amounts of [³H]proline were formed in every cell line studied, with proline ultimately becoming the major radiolabeled product. The putative second product of the hydrolysis of P5, N,N-dimethylvalyl-valyl-N-methylvalyl-proline (P4), had little activity as either an antitubulin or cytotoxic agent. In seven suspension cell lines, the cytotoxicity of tasidotin correlated with total cell uptake of the compound and was probably affected negatively by the extent of degradation of P5 to proline and, presumably, P4. The intracellular enzyme prolyl oligopeptidase probably degrades tasidotin to P5. When CCRF-CEM human leukemia cells were treated with N-benzyloxycarbonylprolylprolinal (BCPP), an inhibitor of prolyl oligopeptidase, there was a 30-fold increase in the IC₅₀ of tasidotin and a marked increase in intracellular [³H]tasidotin. BCPP also caused a 4-fold increase in the IC₅₀ of P5, so the enzyme probably does not convert P5 to P4. Inhibiting degradation of P5 should have led to a decrease in the IC₅₀ obtained for P5 in the presence of BCPP.

[ARTICLES] Opposing Effects of Platelet-Activating Factor and Lyso-Platelet-Activating Factor on Neutrophil and Platelet Activation

12/19/2008 12:00:00 AM

Platelet-activating factor (PAF) is a potent, bioactive phospholipid that acts on multiple cells and tissues through its G protein-coupled receptor (GPCR). PAF is not stored but is rapidly generated via enzymatic acetylation of the precursor 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPAF). The bioactivity of PAF is effectively and tightly regulated by PAF acetylhydrolases, which convert PAF back to lysoPAF. Previous studies report that lysoPAF is an inactive precursor and metabolite of PAF. However, lysoPAF has not been carefully studied in its own context. Here we report that lysoPAF has an opposing effect of PAF in the activation of neutrophils and platelets. Whereas PAF potentiates neutrophil NADPH oxidase activation, lysoPAF dose-dependently inhibits this function. Inhibition by lysoPAF is not affected by the use of a PAF receptor antagonist or genetic deletion of the PAF receptor gene. The mechanism of lysoPAF-mediated inhibition of neutrophils involves an elevation in the intracellular cAMP level, and pharmacological blockade of adenylyl cyclase completely reverses the inhibitory effect of lysoPAF. In addition, lysoPAF increases intracellular cAMP levels in platelets and inhibits thrombin-induced platelet aggregation, which can be reversed by inhibition of protein kinase A. These findings identify lysoPAF as a bioactive lipid with opposing functions of PAF and suggest a novel and intrinsic regulatory mechanism for balance of the potent activity of PAF.

[ARTICLES] The Gq and G12 Families of Heterotrimeric G Proteins Report Functional Selectivity

12/19/2008 12:00:00 AM

Receptors coupled to the G_q and G₁₂ families of heterotrimeric G proteins have surfaced rarely in the context of functional selectivity and always indirectly. We explore here the differential engagement of G_q and G₁₃ (of the G₁₂ family) by the thromboxane A₂ receptor (TP), via agonist-effected [³⁵S]-guanosine 5'-O-(3-thio)triphosphate binding when the G proteins themselves are used as reporters. We find for TP introduced into human embryonic kidney 293 cells and for the receptor expressed normally in human platelets an agonist-selective engagement of G_q versus G₁₃. Pinane thromboxane A₂ (PTA₂) activates G_q in preference to G₁₃, whereas 8-iso-prostaglandin F₂ activates G₁₃ in preference to G_q. 9,11-Dideoxy-9,11-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619), in contrast, exhibits no preference. Reserve of receptor in relation to G protein and of G protein in relation to downstream events is apparent in some instances but does not have a bearing on selectivity. Activation of G proteins by PTA₂ is right-shifted from binding of the ligand to receptor, a manifestation of which is a bimodal action: PTA₂ is an antagonist at low concentrations and an agonist at higher concentrations. We posit two populations of TP, or two intrinsic sites of ligand binding, with selectivity evident not only in terms of the G proteins activated but properties of antagonism versus agonism.

[ARTICLES] Short-Chain Ubiquitination Is Associated with the Degradation Rate of a Cell-Surface-Resident Bile Salt Export Pump (BSEP/ABCB11)

12/19/2008 12:00:00 AM

The reduced expression of the bile salt export pump (BSEP/ABCB11) at the canalicular membrane is associated with cholestasis-induced hepatotoxicity due to the accumulation of bile acids in hepatocytes. We demonstrated previously that 4-phenylbutyrate (4PBA) treatment, a U.S. Food and Drug Administration-approved drug for the treatment of urea cycle disorders, induces the cell-surface expression of BSEP by prolonging the degradation rate of cell-surface-resident BSEP. On the other hand, BSEP mutations, E297G and D482G, found in progressive familial intrahepatic cholestasis type 2 (PFIC2), reduced it by shortening the degradation rate of cell-surface-resident BSEP. Therefore, to help the development of the medical treatment of cholestasis, we investigated the underlying mechanism by which 4PBA and PFIC2-type mutations affect the BSEP degradation from cell surface, focusing on short-chain ubiquitination. In Madin-Darby canine kidney II (MDCK II) cells expressing BSEP and rat canalicular membrane vesicles, the molecular mass of the mature form of BSEP/Bsep shifted from 170 to 190 kDa after ubiquitin modification (molecular mass, 8 kDa). Ubiquitination susceptibility of BSEP/Bsep was reduced in vitro and in vivo by 4PBA treatment and, conversely, was enhanced by BSEP mutations E297G and D482G. Moreover, biotin-labeling studies using MDCK II cells demonstrated that the degradation of cell-surface-resident chimeric protein fusing ubiquitin to BSEP was faster than that of BSEP itself. In conclusion, BSEP/Bsep is modified with two to three ubiquitins, and its ubiquitination is modulated by 4PBA treatment and PFIC2-type mutations. Modulation of short-chain ubiquitination can regulate the change in the degradation rate of cell-surface-resident BSEP by 4PBA treatment and PFIC2-type mutations.

[ARTICLES] Suppression of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-Induced Nitric-Oxide Synthase 2 Expression in Astrocytes by a Novel Diindolylmethane Analog Protects Striatal Neurons against Apoptosis

12/19/2008 12:00:00 AM

The progressive debilitation of motor functions in Parkinson's disease (PD) results from degeneration of dopaminergic neurons within the substantia nigra pars compacta of the midbrain. Long-term inflammatory activation of microglia and astrocytes plays a central role in the progression of PD and is characterized by activation of the nuclear factor-B (NF-B) signaling cascade and subsequent overproduction of inflammatory cytokines and nitric oxide (NO). Suppression of this neuroinflammatory phenotype has received considerable attention as a potential target for chemotherapy, but there are no currently approved drugs that sufficiently address this problem. The data presented here demonstrate the efficacy of a novel anti-inflammatory diindolylmethane class compound, 1,1-bis(3'-indolyl)-1-(p-t-butylphenyl)methane (DIM-C-pPhtBu), in suppressing NF-B-dependent expression of inducible nitric-oxide synthase (NOS2) and NO production in astrocytes exposed to the parkinsonian neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) through a mechanism distinct from that described for the thiazolidinedione-class compound, rosiglitazone. Chromatin immunoprecipitations revealed that micromolar concentrations of DIM-C-pPhtBu prevented association of the p65 subunit of NF-B with enhancer elements in the Nos2 promoter but had little effect on DNA binding of either peroxisome proliferator-activated receptor- (PPAR-) or the nuclear corepressor NCoR2. Treatment with DIM-C-pPhtBu concomitantly suppressed NO production and protein nitration in MPTP-activated astrocytes and completely protected cocultured primary striatal neurons from astrocyte-dependent apoptosis. These data demonstrate the efficacy of DIM-C-pPhtBu in preventing the activation of NF-B-dependent inflammatory genes in primary astrocytes and suggest that this class of compounds may be effective neuroprotective anti-inflammatory agents in vivo.

[ARTICLES] Overlapping Binding Site for the Endogenous Agonist, Small-Molecule Agonists, and Ago-allosteric Modulators on the Ghrelin Receptor

12/19/2008 12:00:00 AM

A library of robust ghrelin receptor mutants with single substitutions at 22 positions in the main ligand-binding pocket was employed to map binding sites for six different agonists: two peptides (the 28-amino-acid octanoylated endogenous ligand ghrelin and the hexapeptide growth hormone secretagogue GHRP-6) plus four nonpeptide agonists—the original benzolactam L-692,429 [3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-([2'-(1H-tetrazol-5-yl) (1,1'-biphenyl)-4-yl]methyl)-1H-1-benzazepin-3(R)-yl)-butanamide], the spiroindoline sulfonamide MK-677 [N-[1(R)-1, 2-dihydro-1-ethanesulfonylspiro-3H-indole-3,4'-piperidin)-1'-yl]carbonyl-2-(phenylmethoxy)-ethyl-2-amino-2-methylpropanamide], and two novel oxindole derivatives, SM-130686 [(+)-6-carbamoyl-3-(2-chlorophenyl)-(2-diethylaminoethyl)-4-trifluoromethyloxindole] and SM-157740 [(±)-6-carbamoyl-3-(2, 4-dichlorophenyl)-(2-diethylaminoethyl)-4-trifluoromethyloxindole)]. The strongest mutational effect with respect to decrease in potency for stimulation of inositol phosphate turnover was for all six agonists the GluIII:09-to-Gln substitution in the extracellular segment of TM-III. Likewise, all six agonists were affected by substitutions of PheVI:16, ArgVI:20, and PheVI:23 on the opposing face of transmembrane domain (TM) VI. Each of the agonists was also affected selectively by specific mutations. The mutational map of the ability of L-692,429 and GHRP-6 to act as allosteric modulators by increasing ghrelin's maximal efficacy overlapped with the common mutational map for agonism but it was not identical with the map for the agonist property of these small-molecule ligands. In molecular models, built over the inactive conformation of rhodopsin, low energy conformations of the nonpeptide agonists could be docked to satisfy many of their mutational hits. It is concluded that although each of the ligands in addition exploits other parts of the receptor, a large, common binding site for both small-molecule agonists—including ago-allosteric modulators—and the endogenous agonist is found on the opposing faces of TM-III and -VI of the ghrelin receptor.

[ARTICLES] Structural Basis of NR2B-Selective Antagonist Recognition by N-Methyl-D-aspartate Receptors

12/19/2008 12:00:00 AM

N-Methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors endowed with unique pharmacological and functional properties. In particular, their high permeability to calcium ions confers on NMDARs a central role in triggering long term changes in synaptic strength. Under excitotoxic pathological conditions, such as those occurring during brain trauma, stroke, or Parkinson's or Huntington's diseases, calcium influx through NMDAR channels can also lead to neuronal injury. This argues for the use of NMDAR antagonists as potential therapeutic agents. To date, the most promising NMDAR antagonists are ifenprodil and derivatives, compounds that act as noncompetitive inhibitors selective for NMDARs containing the NR2B subunit. Recent studies have identified the large N-terminal domain (NTD) of NR2B as the region controlling ifenprodil sensitivity of NMDARs. We present here a detailed characterization of the ifenprodil binding site using both experimental and computational approaches. 3D homology modeling reveals that ifenprodil fits well in a closed cleft conformation of the NRB NTD; however, ifenprodil can adopt either of two possible binding orientations of opposite direction. By studying the effects of cleft mutations, we show that only the orientation in which the phenyl moiety points deep toward the NTD hinge is functionally relevant. Moreover, based on our model, we identify novel NTD NR2B residues that are crucial for conferring ifenprodil sensitivity and provide functional evidence that these residues directly interact with the ifenprodil molecule. This work provides a general insight into the origin of the subunit-selectivity of NMDAR noncompetitive antagonists and offer clues for the discovery of novel NR2B-selective antagonists.

[ARTICLES] Hydrogen Sulfide Inhibits Rotenone-Induced Apoptosis via Preservation of Mitochondrial Function

12/19/2008 12:00:00 AM

Hydrogen sulfide (H₂S) has been proposed as a novel neuromodulator, which plays critical roles in the central nervous system affecting both neurons and glial cells. However, its relationship with neurodegenerative diseases is unexplored. The present study was undertaken to investigate the effects of H₂S on cell injury induced by rotenone, a commonly used toxin in establishing in vivo and in vitro Parkinson's disease (PD) models, in human-derived dopaminergic neuroblastoma cell line (SH-SY5Y). We report here that sodium hydrosulfide (NaHS), an H₂S donor, concentration-dependently suppressed rotenone-induced cellular injury and apoptotic cell death. NaHS also prevented rotenone-induced p38- and c-Jun NH₂-terminal kinase (JNK)-mitogen-activated protein kinase (MAPK) phosphorylation and rotenone-mediated changes in Bcl-2/Bax levels, mitochondrial membrane potential (_m) dissipation, cytochrome c release, caspase-9/3 activation and poly(ADP-ribose) polymerase cleavage. Furthermore, 5-hydroxydecanoate, a selective blocker of mitochondrial ATP-sensitive potassium (mitoK_ATP) channel, attenuated the protective effects of NaHS against rotenone-induced cell apoptosis. Thus, we demonstrated for the first time that H₂S inhibited rotenone-induced cell apoptosis via regulation of mitoK_ATP channel/p38- and JNK-MAPK pathway. Our data suggest that H₂S may have potential therapeutic value for neurodegenerative diseases, such as PD.

[MINIREVIEW] Topology of Class A G Protein-Coupled Receptors: Insights Gained from Crystal Structures of Rhodopsins, Adrenergic and Adenosine Receptors

12/19/2008 12:00:00 AM

Biological membranes are densely packed with membrane proteins that occupy approximately half of their volume. In almost all cases, membrane proteins in the native state lack the higher-order symmetry required for their direct study by diffraction methods. Despite many technical difficulties, numerous crystal structures of detergent solubilized membrane proteins have been determined that illustrate their internal organization. Among such proteins, class A G protein-coupled receptors have become amenable to crystallization and high resolution X-ray diffraction analyses. The derived structures of native and engineered receptors not only provide insights into their molecular arrangements but also furnish a framework for designing and testing potential models of transformation from inactive to active receptor signaling states and for initiating rational drug design.

[PERSPECTIVE] Functional Selectivity of GPCR Ligand Stereoisomers: New Pharmacological Opportunities

12/19/2008 12:00:00 AM

It is now well established that any given ligand for a G-protein-couple receptor (GPCR) does not simply possess a single defined efficacy. Rather, a ligand possesses multiple efficacies, depending on the specific down-stream signal transduction pathway analyzed. This diversity may be based on ligand-specific GPCR conformations and is often referred to as "functional selectivity." It has been known for a century that stereoisomers of catecholamines differ in their potency and, in some systems, also in their efficacy. However, the molecular basis for efficacy differences of GPCR ligand stereoisomers has remained poorly defined. In an elegant study published in this issue of Molecular Pharmacology, Woo et al. (p. 158) show that stereoisomers of the β₂-adrenoceptor selective agonist fenoterol differentially activates G_s- and G_i-proteins in native rat cardiomyocytes. This study is so important because it is the first report to show that even the subtle structural differences within a ligand stereoisomer pair are sufficient to discriminate between GPCR conformations with distinct G-protein coupling properties. The study highlights of how important it is to examine the "more active" (eutomer) and the "less active" (distomer) stereoisomer to understand the mechanisms of action and the cellular effects of GPCR ligands. The study by Woo et al. will ignite a renaissance of the analysis of ligand stereoisomers, using sensitive pharmacological and biophysical assays. The available literature supports the notion that meticulous analysis of ligand stereoisomers is a goldmine for understanding mechanisms of GPCR activation, analysis of signal transduction pathways, development of new therapies for important diseases, and drug safety.

[ARTICLES] An Intracellular Loop 2 Amino Acid Residue Determines Differential Binding of Arrestin to the Dopamine D2 and D3 Receptors

12/19/2008 12:00:00 AM

Dopamine D₂ and D₃ receptors are similar subtypes with distinct interactions with arrestins; the D₃ receptor mediates less agonist-induced translocation of arrestins than the D₂ receptor. The goals of this study were to compare nonphosphorylated arrestin-binding determinants in the second intracellular domain (IC2) of the D₂ and D₃ receptors to identify residues that contribute to the differential binding of arrestin to the subtypes. Arrestin3 bound to glutathione transferase (GST) fusion proteins of the D₂ receptor IC2 more avidly than to the D₃ receptor IC2. Mutagenesis of the fusion proteins identified a residue at the C terminus of IC2, Lys149, that was important for the preferential binding of arrestin3 to D₂-IC2; arrestin binding to D₂-IC2-K149C was greatly decreased compared with wild-type D₂-IC2, whereas binding to the reciprocal mutant D₃-IC2-C147K was enhanced compared with wild-type D₃-IC2. Mutating this lysine in the full-length D₂ receptor to cysteine decreased the ability of the D₂ receptor to mediate agonist-induced arrestin3 translocation to the membrane and decreased agonist-induced receptor internalization in human embryonic kidney 293 cells. The reciprocal mutation in the D₃ receptor increased receptor-mediated translocation of arrestin3 without affecting agonist-induced receptor internalization. G protein-coupled receptor crystal structures suggest that Lys149, at the junction of IC2 and the fourth membrane-spanning helix, has intramolecular interactions that contribute to maintaining an inactive receptor state. It is suggested that the preferential agonist-induced binding of arrestin3 to the D₂ receptor over the D₃ receptor is due in part to Lys149, which could be exposed as a result of receptor activation.

[ARTICLES] A Dopamine D2 Receptor Mutant Capable of G Protein-Mediated Signaling but Deficient in Arrestin Binding

12/19/2008 12:00:00 AM

Arrestins mediate G protein-coupled receptor desensitization, internalization, and signaling. Dopamine D₂ and D₃ receptors have similar structures but distinct characteristics of interaction with arrestins. The goals of this study were to compare arrestin-binding determinants in D₂ and D₃ receptors other than phosphorylation sites and to create a D₂ receptor that is deficient in arrestin binding. We first assessed the ability of purified arrestins to bind to glutathione transferase (GST) fusion proteins containing the receptor third intracellular loops (IC3). Arrestin3 bound to IC3 of both D₂ and D₃ receptors, with the affinity and localization of the binding site indistinguishable between the receptor subtypes. Mutagenesis of the GST-IC3 fusion proteins identified an important determinant of the binding of arrestin3 in the N-terminal region of IC3. Alanine mutations of this determinant (IYIV212-215) in the full-length D₂ receptor generated a signaling-biased receptor with intact ligand binding and G-protein coupling and activation, but deficient in receptor-mediated arrestin3 translocation to the membrane, agonist-induced receptor internalization, and agonist-induced desensitization in human embryonic kidney 293 cells. This mutation also decreased arrestin-dependent activation of extracellular signal-regulated kinases. The finding that nonphosphorylated D₂-IC3 and D₃-IC3 have similar affinity for arrestin is consistent with previous suggestions that the differential effects of D₂ and D₃ receptor activation on membrane translocation of arrestin and receptor internalization are due, at least in part, to differential phosphorylation of the receptors. In addition, these results imply that the sequence IYIV212-215 at the N terminus of IC3 of the D₂ receptor is a key element of the arrestin binding site.

[ARTICLES] 90-kDa Heat Shock Protein Inhibition Abrogates the Topoisomerase I Poison-Induced G2/M Checkpoint in p53-Null Tumor Cells by Depleting Chk1 and Wee1

12/19/2008 12:00:00 AM

The G₂/M cell cycle checkpoint is regulated by a multitude of signaling pathways after genotoxic stress. Herein, we report that treatment with the 90-kDa heat shock protein (Hsp90) molecular chaperone inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) selectively abrogates the G₂/M checkpoint induced by 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan, in p53-null compared with p53-intact HCT116 colon cancer cells. The basis for this selectivity can be explained in part by the lack of p21 induction in p53-null cells. In accord with published results, we could show that treatment with 17AAG resulted in depletion of Chk1, a known Hsp90 client protein. In addition, we observed a time- and dose-dependent decrease in Wee1 kinase level, a negative regulator of mitosis, after 17AAG treatment in gastrointestinal cancer cells. Depletion of Wee1 protein preceded mitotic entry induced by 17AAG, and this decrease could be partially rescued by cotreatment with a proteasome inhibitor. Coimmunoprecipitation experiments showed that Hsp90 and Wee1 interacted in whole cells, and 17AAG treatment decreased the degradative half-life of Wee1, indicating that Wee1 is another Hsp90 client in mammalian cells. Knockdown of Chk1 and Wee1 by short interfering RNA each resulted in abrogation of the G₂/M checkpoint induced by SN-38. The combination of SN-38 and 17AAG was shown to be synergistic in p53-null but not in parental HCT116 cells by median effect/combination index analysis. Taken together, 17AAG specifically inhibits the G₂/M checkpoint in p53-defective cells by down-regulation of two critical checkpoint kinases, Chk1 and Wee1.

[ARTICLES] Thiophenecarboxylate Suppressor of Cyclic Nucleotides Discovered in a Small-Molecule Screen Blocks Toxin-Induced Intestinal Fluid Secretion

12/19/2008 12:00:00 AM

We carried out a "pathway" screen of 50,000 small molecules to identify novel modulators of cAMP signaling. One class of compounds, the 2-(acylamino)-3-thiophenecarboxylates, strongly suppressed cAMP and cGMP in multiple cell lines in response to different agonists acting on G-protein-coupled receptors, adenylyl cyclase, and guanylyl cyclase. The best compounds from structure-activity analysis of 124 analogs, including several synthesized chiral analogs, had and IC₅₀ of <5 µM for suppression of agonist-induced cAMP and cGMP elevation. Measurements of cAMP, cGMP, and downstream signaling in response to various activators/inhibitors suggested that the 2-(acylamino)-3-thiophenecarboxylates function as nonselective phosphodiesterase activators, although it was not determined whether their action on phosphodiesterases is direct or indirect. The 2-(acylamino)-3-thiophenecarboxylates suppressed CFTR-mediated Cl^- current in T84 colonic cells in response to cholera and Escherichia coli (STa) toxins, and prevented intestinal fluid accumulation in a closed-loop mouse model of secretory diarrhea. They also prevented cyst growth in an in vitro renal epithelial cell model of polycystic kidney disease. The 2-(acylamino)-3-thiophenecarboxylates represent the first small-molecule cyclic nucleotide suppressors, whose potential therapeutic indications include secretory diarrheas, polycystic kidney disease, and growth inhibition of cAMP-dependent tumors.

[ARTICLES] Phenylephrine-Induced Cardiomyocyte Injury Is Triggered by Superoxide Generation through Uncoupled Endothelial Nitric-Oxide Synthase and Ameliorated by 3-[2-[4-(3-Chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyindazole (DY-9836), a Novel Calmodulin Antagonist

12/19/2008 12:00:00 AM

The pathophysiological relevance of endothelial nitric-oxide synthase (eNOS)-induced superoxide production in cardiomyocyte injury after prolonged phenylephrine (PE) exposure remains unclear. The aims of this study were to define the mechanism of O₂^. production by uncoupled eNOS and evaluate the therapeutic potential of a novel calmodulin antagonist 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyindazole (DY-9836) to rescue hypertrophied cardiomyocytes from PE-induced injury. In cultured rat cardiomyocytes, prolonged exposure for 96 h to PE led to translocation from membrane to cytosol of eNOS and breakdown of caveolin-3 and dystrophin. When NO and O₂^. production were monitored in PE-treated cells by 4-amino-5-methylamino-2',7'-difluorofluorescein and dihydroethidium, respectively, Ca²⁺-induced NO production elevated by 5.7-fold (p < 0.01) after 48-h PE treatment, and the basal NO concentration markedly elevated (16-fold; p < 0.01) after 96-h PE treatment. On the other hand, the O₂^. generation at 96 h was closely associated with an increased uncoupled eNOS level. Coincubation with DY-9836 (3 µM) during the last 48 h inhibited the aberrant O₂^. generation nearly completely and NO production by 72% (p < 0.01) after 96 h of PE treatment and inhibited the breakdown of caveolin-3/dystrophin in cardiomyocytes. PE-induced apoptosis assessed by TdT-mediated dUTP nick-end labeling staining was also attenuated by DY-9836 treatment. These results suggest that O₂^. generation by uncoupled eNOS probably triggers PE-induced cardiomyocyte injury. Inhibition of abnormal O₂^. and NO generation by DY-9836 treatment represents an attractive therapeutic strategy for PE/hypertrophy-induced cardiomyocyte injury.

[ARTICLES] Imatinib Mesylate (STI571)-Induced Cell Edge Translocation of Kinase-Active and Kinase-Defective Abelson Kinase: Requirements of Myristoylation and src Homology 3 Domain

12/19/2008 12:00:00 AM

4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide methanesulfonate (STI571) is the first successful target-based drug with excellent potency against chronic myelogenous leukemia. Studies on this compound have illuminated potentials and problems of kinase inhibitors in the treatment of cancer. As found in crystal structures, STI571-bound Abelson kinase (abl) is believed to form closed conformation with N-terminal regulatory domains. Here we present evidence of distinct STI571-induced modulation of abl functions using high-resolution live-cell imaging approaches. Within lamellipodia of fibroblast cells, STI571 was found to induce rapid translocation of abl to the lamellipodium tip. Quantitative analysis yielded 0.81 and 1.8 µM for EC₅₀ values of STI571-induced cell edge translocation of abl-KD-green fluorescent protein (GFP) and wild-type abl-GFP, respectively. It also revealed adverse response of drug-resistant abl-T334I to STI571, suggesting that drug binding to abl-GFP triggers translocation. N-myristoylation and the src homology 3 (SH3) domain were required for this translocation, whereas disruption of intramolecular interactions of these motifs enhanced cell-edge association of abl. An intact C-terminal last exon region in abl, but not its F-actin binding, was required for efficient cell-edge translocation. Moreover, single-molecule observation revealed an STI571-induced rapid increase in slow diffusive species of abl in both the tip and the body region of lamellipodia. These results suggest that although activated abl translocates to the cell edge at its open state, STI571 can also bind and lock abl in the open and membrane-tethered conformation as long as the SH3 domain and the C-terminal region are intact. High-resolution imaging can be a powerful tool for elucidating inhibitor modulation of abl functions under intracellular environment.

[ARTICLES] Flow Cytometry-Based Binding Assay for GPR40 (FFAR1; Free Fatty Acid Receptor 1)

12/19/2008 12:00:00 AM

GPR40 is a G protein-coupled receptor (GPCR) whose endogenous ligands have recently been identified as medium- and long-chain free fatty acids (FFAs), and it is thought to play an important role in insulin release. Despite recent research efforts, much still remains unclear in our understanding of its pharmacology, mainly because the receptor-ligand interaction has not been analyzed directly. To study the pharmacology of GPR40 in a more direct fashion, we developed a flow cytometry-based binding assay. FLAG-tagged GPR40 protein was expressed in Sf9 cells, solubilized, immobilized on immunomagnetic beads, and labeled with the fluorescent probe C1-BODIPY-C12. Flow cytometry analysis showed that C1-BODIPY-C12 specifically labels a single class of binding site in a saturable and reversible manner with an apparent dissociation constant of ~3 µM. The FFAs that activate GPR40 competed with C1-BODIPY-C12 binding; thus, medium- to long-chain FFAs could compete, whereas short-chain FFAs and methyl linoleate had no inhibitory effect. Furthermore, ligands that are known to activate GPR40 competed for binding in a concentration-dependent manner. All the ligands that inhibited the binding promoted phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in human embryonic kidney (HEK) 293 cells that expressed GPR40 and [Ca²⁺]_i responses in mouse insulinoma (MIN6) cells that natively express GPR40; however, pioglitazone, a thiazolidinedione that failed to compete for the binding, did not activate ERK or [Ca²⁺]_i response. This study showed that a flow cytometry-based binding assay can successfully identify direct interactions between GPR40 and its ligands. This approach would be of value in studying the pharmacology of GPCRs.

[ARTICLES] Inhibition of P-Glycoprotein-Mediated Paclitaxel Resistance by Reversibly Linked Quinine Homodimers

12/19/2008 12:00:00 AM

P-glycoprotein (P-gp), an ATP-dependent drug efflux pump, has been implicated in multidrug resistance of several cancers as a result of its overexpression. In this work, rationally designed second-generation P-gp inhibitors are disclosed, based on dimerized versions of the substrates quinine and quinidine. These dimeric agents include reversible tethers with a built-in clearance mechanism. The designed agents were potent inhibitors of rhodamine 123 efflux in cultured cancer cell lines that display high levels of P-gp expression at the cell surface and in transfected cells expressing P-gp. The quinine homodimer Q2, which was tethered by reversible ester bonds, was particularly potent (IC₅₀ 1.7 µM). Further studies revealed that Q2 inhibited the efflux of a range of fluorescent substrates (rhodamine 123, doxorubicin, mitoxantrone, and BODIPY-FL-prazosin) from MCF-7/DX1 cells. The reversibility of the tether was confirmed in experiments showing that Q2 was readily hydrolyzed by esterases in vitro (t_1/2 20 h) while demonstrating high resistance to nonenzymatic hydrolysis in cell culture media (t_1/2 21 days). Specific inhibition of [¹²⁵I]iodoarylazidoprazosin binding to P-gp by Q2 verified that the bivalent agent interacted specifically with the drug binding site(s) of P-gp. Q2 was also an inhibitor of verapamil-stimulated ATPase activity. In addition, low concentrations of Q2 stimulated basal P-gp ATPase levels. Finally, Q2 was shown to inhibit the transport of radiolabeled paclitaxel (Taxol) in MCF-7/DX1 cells, and it completely reversed the P-gp-mediated paclitaxel resistance phenotype.