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Biochemical Pharmacology (v.71, #4)

Editorial Advisory Board (pp. iii).

Neuronal and astrocytic shuttle mechanisms for cytosolic-mitochondrial transfer of reducing equivalents: Current evidence and pharmacological tools by Mary C. McKenna; Helle S. Waagepetersen; Arne Schousboe; Ursula Sonnewald (pp. 399-407).

The malate–aspartate shuttle and the glycerol phosphate shuttle act to transfer reducing equivalents from NADH in the cytosol to the mitochondria since the inner mitochondrial membrane is impermeable to NADH and NAD⁺. This transfer of reducing equivalents is essential for maintaining a favorable NAD⁺/NADH ratio required for the oxidative metabolism of glucose and synthesis of neurotransmitters in brain. There is evidence that both the malate–aspartate shuttle and glycerol phosphate shuttle function in brain; however, there is controversy about the relative importance and cellular localization of these shuttles. The malate–aspartate shuttle is considered the most important shuttle in brain. It is particularly important in neurons and may be extremely low, or even non-existent in brain astrocytes. Several studies provide evidence of glycerol phosphate shuttle activity in brain cells; however, the activity of this shuttle in brain has been questioned. A number of pharmacological tools, including aminooxyacetic acid, β-methyleneaspartate, phenylsuccinate, and 3-nitropropionic acid, have been used to inhibit the four enzymes and two carrier proteins that participate in the malate–aspartate shuttle. Although no drugs completely inhibit the glycerol phosphate shuttle, evidence for the existence of this shuttle is provided by studies using drugs to inhibit the malate–aspartate shuttle. This report evaluates the evidence for each shuttle in brain cells and the drugs that can be used as pharmacological tools to study these shuttles.

Keywords: Neuronal and astrocytic shuttle mechanisms; AOAA; BMA; 3-NPA; Phenylsuccinate; aralar1; Aspartate aminotransferase; Malate dehydrogenase; Malate–aspartate shuttle; Glycerol 3-phosphate shuttle; Malate–α-ketoglutarate carrier; Neurons; Astrocytes; Synaptosomes; Energy metabolism; Aspartate–glutamate carrier; AGC1

Anti-tumour activity in non-small cell lung cancer models and toxicity profiles for novel ruthenium(II) based organo-metallic compounds by S.M. Guichard; R. Else; E. Reid; B. Zeitlin; R. Aird; M. Muir; M. Dodds; H. Fiebig; P.J. Sadler; D.I. Jodrell (pp. 408-415).

Novel ruthenium(II) organo-metallic compounds are active in ovarian cancer models [Aird RE, Cummings J, Ritchie AA, Muir M, Morris RE, Chen H, et al. In vitro and in vivo activity and cross resistance profiles of novel ruthenium(II) organometallic arene complexes in human ovarian cancer. Br J Cancer 2002;86(10):1652–7]. [(η⁶-C₆H₅C₆H₅)Ru(en)Cl]⁺ (as a PF₆ salt, where en=ethylenediamine (RM175)) has been evaluated in a 13-cell line panel. Particular sensitivity (∼10-fold lower than mean IC₅₀) was noted in breast cancer and non-small cell lung cancer cell lines. In addition, IC₅₀ in the A549 was 2μM and RM175 (25mgkg⁻¹, days 1 and 5, i.p.) caused a significant ( p=0.004) growth delay in a xenograft model. HC11 [(η⁶-tetrahydroanthracene)Ru(en)Cl]PF₆ was more potent in the A549 cell line (IC₅₀ 0.5μM). HC11 (25mgkg⁻¹, days 1, 8 and 15, i.p.) was also active in vivo. Following RM175 25mgkg⁻¹, days 1 and 5, and 15mgkg⁻¹, days 1–5, HC11 25 and 40mgkg⁻¹, day 1, elevated alanine transaminase levels were detected, suggesting hepatotoxicity. No changes were observed in kidney or haematological parameters. In liver sections, multi-focal hepatic necrosis was seen, becoming confluent at high doses of HC11. In vitro studies confirmed that HC11 was more toxic than RM175 to fresh human hepatocytes and equitoxic to mithramycin. Liver toxicity may be related to the arene ligand and modification may reduce the potential for hepatic toxicity, while maintaining the anti-tumour activity seen.

Keywords: Ruthenium(II) organo-metallic complex; Lung cancer; Hepatotoxicity

Overcoming methotrexate resistance in breast cancer tumour cells by the use of a new cell-penetrating peptide by Maria Lindgren; Katri Rosenthal-Aizman; K�lliki Saar; Emel�a Eir�ksd�ttir; Yang Jiang; Meeri Sassian; Pernilla �stlund; Mattias H�llbrink; �lo Langel (pp. 416-425).

Resistance to chemotherapy limits the effectiveness of anti-cancer drug treatment. Here, we present a new approach to overcome the setback of drug resistance by designing a conjugate of a cell-penetrating peptide and the cytostatic agent methotrexate (MTX). Two different peptides, YTA2 and YTA4, were designed and their intracellular delivery efficiency was characterized by fluorescence microscopy and quantified by fluorometry. MTX was conjugated to the transport peptides and the ability of the peptide–MTX conjugates to inhibit dihydrofolate reductase, the target enzyme of MTX, was found to be 15 and 20 times less potent than MTX. In addition, in vitro studies were performed in a drug resistant cell model using the 100-fold MTX resistant breast cancer cells MDA-MB-231. At a concentration of 1μM, the peptide–MTX conjugates were shown to overcome MTX resistance and kill the cells more efficiently than MTX alone. Estimated EC₅₀'s were determined for MTX, MTX-YTA2 and YTA2 to be 18.5, 3.8 and 20μM, respectively. In summary, cell-penetrating peptide conjugation of MTX is a new way of increasing delivery, and thereby, the potency of already well-characterized therapeutic molecules into drug resistant tumour cells.

Keywords: Abbreviations; Ac; acetylated; APA; 4-amino-4-deoxy-; N; ¹⁰; -methyl pteroic acid; BOP; benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; CPP; cell-penetrating peptide; DHFR; dehydrate folate reductase; LDH; lactate dehydrogenase; MALDI; matrix assisted laser desorption ionization; MS; mass spectrometry; MTX; methotrexate; RP-HPLC; reversed phase high performance liquid chromatography; TBTU; 1H-benzotriazole-1-yl)-; N; ,; N; ,; N; ′,; N; ′-tetramethyluronium tetrafluoroborate; TFA; trifluoro acetic acidCell-penetrating peptide; Drug delivery; Methotrexate; MDA-MB-231 cells; Drug resistance; Drug delivery

Anticancer activity of the lanthanum compound [tris(1,10-phenanthroline)lanthanum(III)]trithiocyanate (KP772; FFC24) by Petra Heffeter; Michael A. Jakupec; Wilfried K�rner; Stefan Wild; Nikolai Graf von Keyserlingk; Leonilla Elbling; Haralabos Zorbas; Alla Korynevska; Siegfried Knasm�ller; Hedwig Sutterl�ty; Michael Micksche; Bernhard K. Keppler; Walter Berger (pp. 426-440).

Aim of this study was to investigate the anticancer properties of the new lanthanum compound [tris(1,10-phenanthroline)lanthanum(III)]trithiocyanate (KP772; FFC24). In vitro, growth inhibition by KP772 was comparable for >60 tumour cell models with IC₅₀ values generally in the low μM range. KP772 induced tumour cell apoptosis indicated by chromatin condensation, caspase substrate cleavage and mitochondrial membrane depolarisation. DNA is unlikely to represent the primary molecular target of KP772, as no significant interaction or damage of DNA was detectable both in vitro and in living cells. Moreover, we found no evidence for induction of radical species. In contrast, KP772 potently inhibited DNA synthesis paralleled by a massive block of cell cycle in G₀/G₁ phase and a selective decrease of cyclin B₁. Although treatment with KP772 induced expression of p53 and p21^Waf1, transfection of wild-type p53 into knock-out cells only marginally enhanced the cytostatic activity of KP772. In vivo, the anticancer activity of KP772 against human DLD-1 colon carcinoma xenografts was comparable to that of cisplatin and methotrexate at doses not causing significant adverse effects. With regard to toxicity, the LD₅₀ and no-observed-adverse-effect levels (NOAEL) of KP772 in Sprague–Dawley rats were 21.6 and 7.5mg/kg, in outbred albino mice 62 and 10mg/kg, respectively. In summary, KP772 exerts anticancer activity via potent induction of cell cycle arrest and/or apoptosis and has promising in vivo anticancer activity against a human colon cancer xenograft. Together, these data suggest further development of KP772 as a new anticancer metal-drug.

Keywords: Abbreviations; CDDP; cisplatin; ICP-MS; inductively coupled plasma mass spectroscopy; KP772; [tris(1,10-phenanthroline)lanthanum(III)]trithiocyanate; MTD; maximum tolerated dose; MTX; methotrexate; NCI; National Cancer Institute; 1,10-phen; 1,10-phenanthroline; PI; propidium iodide; ROS; reactive oxygen species; TMAH; tetra methyl ammonium hydroxideLanthanum; 1,10-Phenanthroline; Cell cycle arrest; Anticancer activity; Apoptosis

Critical role of PI3-kinase/Akt activation in the PARP inhibitor induced heart function recovery during ischemia–reperfusion by Krisztina Kovacs; Ambrus Toth; Peter Deres; Tamas Kalai; Kalman Hideg; Ferenc Gallyas Jr.; Balazs Sumegi (pp. 441-452).

Poly(ADP-ribose) polymerase (PARP) inhibitors protect hearts from ischemia–reperfusion (IR)-induced damages by limiting nicotinamide adenine dinucleotide (NAD⁺) and ATP depletion, and by other, not yet elucidated mechanisms. Our preliminary data suggested that PARP catalyzed ADP-ribosylations may affect signaling pathways in cardiomyocytes. To clarify this possibility, we studied the effect of a well-characterized (4-hydroxyquinazoline) and a novel (carboxaminobenzimidazol-derivative) PARP inhibitor on the activation of phosphatidylinositol-3-kinase (PI3-kinase)/Akt pathway in Langendorff-perfused hearts. PARP inhibitors promoted the restoration of myocardial energy metabolism (assessed by³¹P nuclear magnetic resonance spectroscopy) and cardiac function compared to untreated hearts. PARP inhibitors also attenuated the infarct size and reduced the IR-induced lipid peroxidation, protein oxidation and total peroxide concentration. Moreover, PARP inhibitors facilitated Akt phosphorylation and activation, as well as the phosphorylation of its downstream target glycogen synthase kinase-3β (GSK-3β) in normoxia and, more robustly, during IR. Blocking PI3-kinase by wortmannin or LY294002 reduced the PARP inhibitor-elicited robust Akt and GSK-3β phosphorylation upon ischemia–reperfusion, and significantly diminished the recovery of ATP and creatine phosphate showing the importance of Akt activation in the recovery of energy metabolism. In addition, inhibition of PI3-kinase/Akt pathway decreased the protective effect of PARP inhibitors on infarct size and the recovery of heart functions. All these data suggest that contrary to the original view, which considered preservation of NAD⁺ and consequently ATP pools as the exclusive underlying mechanism for the cytoprotective effect of PARP inhibitors, the activation of PI3-kinase/Akt pathway and related processes are at least equally important in the cardioprotective effects of PARP inhibitors during ischemia–reperfusion.

Keywords: Ischemia–reperfusion; PARP inhibitors; ROS; Myocardial energy metabolism; Signal transduction; PI3-kinase/AktAbbreviations; GSK-3β; glycogen synthase kinase-3β; IR; ischemia–reperfusion; NAD; ⁺; nicotinamide adenine dinucleotide; PARP; poly(ADP-ribose) polymerase; PI3-kinase; phosphatidylinositol-3-kinase; ROS; reactive oxygen species; TBARS; thiobarbituric acid reactive substances

Different roles of liver X receptor α and β in lipid metabolism: Effects of an α-selective and a dual agonist in mice deficient in each subtype by Erik G. Lund; Laurence B. Peterson; Alan D. Adams; My-Hanh N. Lam; Charlotte A. Burton; Jayne Chin; Qiu Guo; Shaei Huang; Melanie Latham; Jacqueline C. Lopez; John G. Menke; Denise P. Milot; Lyndon J. Mitnaul; Sandra E. Rex-Rabe; Raymond L. Rosa; Jenny Y. Tian; Samuel D. Wright; Carl P. Sparrow (pp. 453-463).

Liver X receptor (LXR) α and LXRβ are closely related nuclear receptors that respond to elevated levels of intracellular cholesterol by enhancing transcription of genes that control cholesterol efflux and fatty acid biosynthesis. The consequences of inactivation of either LXR isoform have been thoroughly studied, as have the effects of simultaneous activation of both LXRα and LXRβ by synthetic compounds. We here describe the effects of selective activation of LXRα or LXRβ on lipid metabolism. This was accomplished by treating mice genetically deficient in either LXRα or LXRβ with an agonist with equal potency for both isoforms (Compound B) or a synthetic agonist selective for LXRα (Compound A). We also determined the effect of these agonists on gene expression and cholesterol efflux in peritoneal macrophages derived from wild-type and knockout mice. Both compounds raised HDL-cholesterol and increased liver triglycerides in wild-type mice; in contrast, in mice deficient in LXRα, Compound B increased HDL-cholesterol but did not cause hepatic steatosis. Compound B induced ATP-binding cassette transporter (ABC) A1 expression and stimulated cholesterol efflux in macrophages from both LXRα and LXRβ-deficient mice. Our data lend further experimental support to the hypothesis that LXRβ-selective agonists may raise HDL-cholesterol and stimulate macrophage cholesterol efflux without causing liver triglyceride accumulation.

Keywords: Nuclear receptor; ATP-binding cassette transporter A1; Sterol regulatory element-binding protein-1c; High density lipoprotein; Cholesterol; Triglycerides; Macrophages

Conformational induction is the key process for activation of the AT₁ receptor by James Ellis; Philip Warburton; Dan Donnelly; Anthony J. Balmforth (pp. 464-471).

It is currently unclear whether activation of the AT₁ receptor by agonists involves conformational selection or induction. We evaluated the pharmacological properties of wild type and N111G CAM human AT₁ receptors stably expressed in HEK293 cells. Although [Sar¹]-Ang II and Ang IV were full agonists at both receptors, the potency of Ang IV was 280-fold lower at the wild type receptor. [Sar¹, Ile⁸]-Ang II was only a full agonist at the N111G CAM AT₁ receptor. [Sar¹]-Ang II and [Sar¹, Ile⁸]-Ang II displayed similar high affinity binding to both receptors. In contrast, Ang IV displayed low affinity binding to the wild type and high affinity binding to the N111G CAM AT₁ receptor. Based on these observations we provide strong evidence that conformational induction is the key process for activation of the AT₁ receptor. Only by the creation of CAMs can conformational selection be envisaged to take place.

Keywords: AT; ₁; receptor; Constitutively active; Extended ternary complex model; Conformational selection; Conformational induction

The effects of prostaglandin F_2α treatment on peripheral-type benzodiazepine receptors in the ovary and uterus during pseudopregnancy of rats by Shalom Bar-Ami; Nachum Bendel; Svetlana Leschiner; Evgeny Levin; Leo Veenman; Moshe Gavish (pp. 472-478).

A previous study by us indicated that peripheral-type benzodiazepine receptor (PBR) density may be increased in the ovaries and uterus of pregnant rats (Weizman R, Dagan E, Snyder SH, Gavish M. Impact of pregnancy and lactation on GABA_A receptor and central-type and peripheral-type benzodiazepine receptors. Brain Res 1997;752:7–14). In the present study, the effects of prostaglandin F_2α (PGF_2α) on PBR density in the ovary and uterus of pseudopregnant rats were assayed. Pseudopregnancy was induced on day 29 post-partum (PP) by s.c. injection of 50IU pregnant mare serum gonadotropin (PMSG) and 3 days later by s.c. injection of 20IU human chorionic gonadotropin (hCG). PBR ligand binding density was assayed with the specific PBR ligand [³H]PK 11195. A two-fold increase in ovarian PBR density was observed 2 days after hCG administration compared with vehicle control rats and this effect was maintained for 3 weeks. In the uterus, a three-fold increase in PBR density was observed and this increase was maintained for 1 week after hCG administration. Pseudopregnancy did not appear to affect renal PBR density or affinity. Treatment with PGF_2α, which causes luteolysis, resulted in an approximately 50% reduction of PBR density in the ovaries of pseudopregnant rats at day 53 PP compared to pseudopregnant control rats. Treatment with indomethacin, which prevents the formation of PGF_2α, caused the PBR density in the uterus of pseudopregnant rats at day 53 PP to be twice as high as in pseudopregnant control rats. All the above treatments did not affect the affinity of [³H]PK 11195 to ovarian and uterine PBR. These data suggest that PBR density in corpora lutea and uterus during pseudopregnancy is regulated by PGF_2α.

Keywords: PBR; Gonadotropins; PGF; _2α; Uterus; Ovary; Progesterone; Pseudopregnancy; [; ³; H]PK 11195

ZD6474 inhibits proliferation and invasion of human hepatocellular carcinoma cells by Gianluigi Giannelli; Amalia Azzariti; Concetta Sgarra; Letizia Porcelli; Salvatore Antonaci; Angelo Paradiso (pp. 479-485).

Hepatocellular carcinoma (HCC) is characterized by hypervascularization, neoangiogenesis formation and blood vessel invasion. Recently, it has been demonstrated that an inhibitor of the vascular endothelial growth factor (VEGF) receptor, ZD6474, may directly inhibit the growth of tumor cells. ZD6474 effectiveness was investigated on cell growth, apoptosis, adhesion, migration and invasion and related to the drug-dependent modulation of main molecular targets on HCC cells. ZD6474 inhibited HCC cell proliferation, however, such effect was reverted by Laminin-5 (Ln-5) but not by other extracellular matrix proteins (ECM). ZD6474 also inhibited HCC cell adhesion, migration and invasion, whereas the simultaneous treatment with the drug and Ln-5 strongly recovered those effects. Under the same experimental conditions, ZD6474 inhibited the expression of phosphorylated EGFR in all cell lines while the effect on p-Erk1/2 was dependent on cellular invasive characteristics. Nonetheless, co-incubation with Ln-5 completely recovered this effect. Our results support the hypothesis that ZD6474 could represent an interesting therapeutic opportunity for patients with HCC scarcely expressing the ECM protein, Ln-5.

Keywords: Hepatocellular carcinoma; Laminin-5; ZD6474; Extracellular matrix proteins; Vascular endothelial growth factor receptor; Biological therapies; Invasion; Proliferation

Modulation of intestinal barrier properties by miltefosine by C�cile Menez; Marion Buyse; H�l�ne Chacun; Robert Farinotti; Gillian Barratt (pp. 486-496).

Miltefosine (hexadecylphosphocholine, HePC) is the first effective oral agent for the treatment of visceral leishmaniasis. This study aimed to determine whether this oral administration alters the integrity and transport capacities of the intestinal barrier. The objectives of this study were: (i) to evaluate the cytotoxicity of HePC, (ii) to investigate the effects of HePC on paracellular and transcellular transport and (iii) to investigate the influence of HePC on three major transporters of the intestinal barrier, namely, P-glycoprotein, the human intestinal peptide transporter (PepT-1) and the monocarboxylic acid transporter (MCT-1) in Caco-2 cell monolayers, used as an in vitro model of the human intestinal barrier. We show that HePC reduced the transepithelial electrical resistance and increasedd-[¹⁴C]mannitol permeability in a dose-dependent manner but had no effect on [³H]testosterone permeability, demonstrating that HePC treatment enhances paracellular permeability via an opening of the tight junction complex without affecting the transcellular route. Morphological studies using confocal fluorescence microscopy showed no perturbation of the normal distribution of ZO-1, occludin or E-cadherin but revealed a redistribution of the tight junction-associated protein claudin-1 and the perijunctional actin after incubation with HePC. Finally, HePC was found to inhibit the intestinal P-glycoprotein in the Caco-2 cell model after a single short exposure. These results suggest that HePC could modify the oral bioavailability of other therapeutic compounds absorbed via the paracellular route or which are substrates of the intestinal P-glycoprotein.

Keywords: Miltefosine; Hexadecylphosphocholine; Caco-2; Tight junctions; Paracellular permeability; P-glycoprotein

Gene expression profiles of the rat brain both immediately and 3 months following acute sarin exposure by Tirupapuliyur V. Damodaran; Anand G. Patel; Stephen T. Greenfield; Holly K. Dressman; Simon M. Lin; Mohamed B. Abou-Donia (pp. 497-520).

We have studied sarin-induced global gene expression patterns at an early time point (15min; 0.5�LD₅₀) and a later time point (3 months; 1�LD₅₀) using Affymetrix: Rat Neurobiology U34 chips in male, Sprague-Dawley rats and have identified a total of 65 (early) and 38 (late) genes showing statistically significant alterations from control levels at 15min and 3 months, respectively. At the early time point, those that are classified as ion channel, cytoskeletal and cell adhesion molecules, in addition to neuropeptides and their receptors predominated over all other groups. The other groups included: cholinergic signaling, calcium channel and binding proteins, transporters, chemokines, GABAnergic, glutamatergic, aspartate, catecholaminergic, nitric oxide synthase, purinergic, and serotonergic signaling molecules. At the late time point, genes that are classified as calcium channel and binding proteins, cytoskeletal and cell adhesion molecules and GABAnergic signaling molecules were most prominent. Seven molecules (Ania-9, Arrb-1, CX-3C, Gabab-1d, Nos-2a, Nrxn-1b, PDE2) were identified that showed altered persistent expression in both time points. Selected genes from each of these time points were further validated using semi quantitative RT-PCR approaches. Some of the genes that were identified in the present study have been shown to be involved in organophosphate-induced neurotoxicity by both other groups as well as ours. Principal component analysis (PCA) of the expression data from both time points was used for comparative analysis of the gene expression, which indicated that the changes in gene expression were a function of dose and time of euthanasia after the treatment. Our model also predicts that besides dose and duration of post-treatment period, age and possibly other factors may be playing important roles in the regulation of pathways, leading to the neurotoxicity.

Keywords: Abbreviations; ACh; acetylcholine; AChE; acetylcholinesterase; ATP; adenosine tri-phosphate; Bfgf; basic fibroblastic growth factor; BBB; blood brain barrier; CREB; cAMP-response element binding protein; CaM KinaseII; Ca; ²⁺; /calmodulin-dependent protein kinase II; camp; cyclic AMP; CNS; central nervous system; CRE; cyclic-AMP responsive element; DEPC; diethylpyrocarbonate; DFP; diisopropylphosphorofluoridate; ERK; extra-cellular signal-regulated kinase; EST; expressed sequence tag; FSH; follicle stimulating hormone; GFAP; glial fibrillary acidic protein; IEG; immediate early gene; JNK; Jun-C N-terminal Kinase; MSK; mitogen and stress activated kinase; mAChE; muscarinic acetylcholine receptor; MAPK; mitogen activated protein kinase; nAChE; nicotinic acetylcholine receptor; NGF; neural growth factor; NF; neurofilament(s); NO; nitric oxide; OP; organophosphates; OPICN; organophosphorus ester-induced chronic neurotoxicity; OPIDN; organophosphorus ester-induced delayed neurotoxicity; p-CREB; phospho-CREB; PCA; principle component analysis; PCD; programmed cell death; PKA; protein kinase A; RT-PCR; reverse transcriptase-polymerase chain reaction; SAGE; serial analysis of gene expression; SAPK; stress activated protein kinase; SRE; serum response elementSarin; Organophosphates; Gene expression; Microarray; Cholinergic system; Acetylcholinesterase

Distinct functional profiles of aripiprazole and olanzapine at RNA edited human 5-HT_2C receptor isoforms by Jean Y. Zhang; Dianne M. Kowal; Stanley P. Nawoschik; Zhuangwei Lou; John Dunlop (pp. 521-529).

In this study we have functionally characterized aripiprazole (OPC-14597; 7-(4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butyloxy-3,4-dihydro-2-(1H)-quinolinone), the prototype of a new generation antipsychotic drug termed dopamine–serotonin-system stabilizer, in cells expressing 5-hydroxytryptamine2 (5-HT₂) receptor subtypes in comparison with olanzapine. In Chinese hamster ovary (CHO) cells stably expressing 5-HT₂ receptors, aripiprazole displayed a dual agonist/antagonist profile for 5-HT_2C receptor (VNI isoform) mediated calcium signaling (EC₅₀ 1070nM, IC₅₀ 281nM). It exhibited no appreciable 5-HT_2A or 5-HT_2B agonism, whereas it antagonized 5-HT-stimulated calcium increase at either 5-HT_2A or 5-HT_2B receptor expressed in CHO cells (IC₅₀s of 369 and 0.46nM, respectively). In comparison, olanzapine was devoid of agonism but was an antagonist at all three subtypes, with a potency rank order of 5-HT_2A (IC₅₀, 2.5nM)>5-HT_2B (47nM)>5-HT_2C (69nM). In human embryonic kidney (HEK) cells transiently expressing 5-HT_2C receptor isoforms, aripiprazole exhibited full agonism at the unedited INI, but partial agonism at the partially edited VNI and fully edited VSV isoforms (EC_5Os of 571, 1086 and 2099nM, respectively). A partial antagonism was also observed for aripiprazole at the two edited isoforms (IC₅₀s of 1138 and 1000nM, respectively). In contrast, while lacking agonist activity at the VNI and VSV, olanzapine showed inverse agonism at the INI isoform (IC₅₀ 594nM), reaching a maximal attenuation of 20%. In addition, olanzapine was a full antagonist at all three isoforms, with a rank order of potency of VNI (IC₅₀, 79nM)>VSV (101nM)>INI (3856nM). The modest 5-HT_2A antagonism and 5-HT_2C partial agonism, along with reported D₂ and 5-HT_1A partial agonism, may allow aripiprazole to stabilize the disturbed dopamine–serotonin interplay in schizophrenia with a moderate yet adequate pharmacological intervention. 5-HT_2C agonism may also underlie the minimal weight gain seen with aripiprazole.

Keywords: Aripiprazole; Schizophrenia; Antipsychotics; 5-HT2 receptors; 5-HT2C isoforms; Functional activity

Potencies of topical glucocorticoids to mediate genomic and nongenomic effects on human peripheral blood mononuclear cells by Lydia Naumann; Eugen Feist; Rainer H. Straub; Gerd-R�diger Burmester; Frank Buttgereit (pp. 530-539).

Several different genomic and nongenomic mechanisms are known to mediate the important anti-inflammatory and immunomodulatory effects of glucocorticoids (GC). Genomic effects are the most important while the clinical relevance of nongenomic actions is still a matter of debate. We therefore investigated whether beclometasone and clobetasol are particularly suitable for topical application because of their specific spectrum of genomic and nongenomic actions.For these purposes we compared effects on oxygen consumption as measured with a Clark electrode (nonspecific nongenomic glucocorticoid effects), on interleukin-6 synthesis by means of ELISA (genomic effects) and on apoptosis using flow cytometry (nongenomic and genomic effects) in quiescent and mitogen-stimulated PBMC.Beclometasone and clobetasol indeed had stronger effects on the oxygen consumption of quiescent and stimulated cells at lower concentrations (10⁻¹⁰ and 10⁻⁸M) but were less potent at higher concentrations (10⁻⁵ and 10⁻⁴M) in comparison with dexamethasone. Also in terms of genomic potency, topical GC were more effective than dexamethasone at 10⁻¹⁰ and 10⁻⁸M but gave similar results at higher concentrations. The ability of all three GC to induce apoptosis was found to be concentration-dependent and similar at concentrations between 10⁻⁸ and 10⁻⁵M. But, compared with 10⁻⁴M dexamethasone, topical GC at 10⁻⁴M were significantly more effective at inducing apoptosis in both PBMC and Jurkat T-cells.These results show that topical GC have different concentration – (genomic/nongenomic) effect – ratios compared with dexamethasone: besides to the well-known genomic effects there are also significant nongenomic effects of topical glucocorticoids that already at low concentrations might be more therapeutically relevant in certain clinical conditions than currently assumed.

Keywords: Abbreviations; GC; glucocorticoid; cGR; cytosolic glucocorticoid receptor; mGR; membrane-bound glucocorticoid receptor; Con A; concanavalin A; PHA; phytohaemagglutinin; IL; interleukin; mab; monoclonal antibody; PBMC; peripheral blood mononuclear cellsHuman peripheral blood mononuclear cells; Systemic and topical glucocorticoids; Energy metabolism; Apoptosis; IL-6; Genomic and nongenomic glucocorticoid effects

Structure activity and molecular modeling analyses of ribose- and base-modified uridine 5′-triphosphate analogues at the human P2Y₂ and P2Y₄ receptors by Kenneth A. Jacobson; Stefano Costanzi; Andrei A. Ivanov; Susanna Tchilibon; Pedro Besada; Zhan-Guo Gao; Savitri Maddileti; T. Kendall Harden (pp. 540-549).

With the long-term goal of developing receptor subtype-selective high affinity agonists for the uracil nucleotide-activated P2Y receptors we have carried out a series of structure activity and molecular modeling studies of the human P2Y₂ and P2Y₄ receptors. UTP analogues with substitutions in the 2′-position of the ribose moiety retained capacity to activate both P2Y₂ and P2Y₄ receptors. Certain of these analogues were equieffective for activation of both receptors whereas 2′-amino-2′-deoxy-UTP exhibited higher potency for the P2Y₂ receptor and 2′-azido-UTP exhibited higher potency for the P2Y₄ receptor. 4-Thio substitution of the uracil base resulted in a UTP analogue with increased potency relative to UTP for activation of both the P2Y₂ and P2Y₄ receptors. In contrast, 2-thio substitution and halo- or alkyl substitution in the 5-position of the uracil base resulted in molecules that were 3–30-fold more potent at the P2Y₂ receptor than P2Y₄ receptor. 6-Aza-UTP was a P2Y₂ receptor agonist that exhibited no activity at the P2Y₄ receptor. Stereoisomers of UTPαS and 2′-deoxy-UTPαS were more potent at the P2Y₂ than P2Y₄ receptor, and the R-configuration was favored at both receptors. Molecular docking studies revealed that the binding mode of UTP is similar for both the P2Y₂ and P2Y₄ receptor binding pockets with the most prominent dissimilarities of the two receptors located in the second transmembrane domain (V90 in the P2Y₂ receptor and I92 in the P2Y₄ receptor) and the second extracellular loop (T182 in the P2Y₂ receptor and L184 in the P2Y₄ receptor). In summary, this work reveals substitutions in UTP that differentially affect agonist activity at P2Y₂ versus P2Y₄ receptors and in combination with molecular modeling studies should lead to chemical synthesis of new receptor subtype-selective drugs.

Keywords: Abbreviations; PLC; phospholipase C; HEPES; N; -(2-hydroxyethyl)-piperazine-; N; ′-2-ethanesulfonic acid; ATP; adenosine 5′-triphosphate; CTP; cytidine 5′-triphosphate; GTP; guanosine 5′-triphosphate; TM; transmembrane domain; TBAP; tetrabutylammonium dihydrogen phosphate; TEAA; triethylammonium acetate; UDP; uridine 5′-diphosphate; UTP; uridine 5′-triphosphateStructure activity relationship; G protein-coupled receptor; Nucleotides; Phospholipase C; Pyrimidines; Homology modeling

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Biochemical Pharmacology (v.71, #4)