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Biochemical Pharmacology (v.73, #2)

Editorial Advisory Board (pp. iii).

Prostanoids in nociception and pain by Hanns Ulrich Zeilhofer (pp. 165-174).

Prostaglandins are lipid mediators produced by cyclooxygenases from arachidonic acid, which serve pivotal functions in inflammation and pain. Inhibition of their production is the major analgesic mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs)—but also the source of most of their unwanted effects. While the development of selective inhibitors of inducible cyclooxygenase (COX)-2 (so called coxibs) has greatly reduced gastrointestinal side effects, the recent disappointment about a potential cardiovascular toxicity of COX-2-selective inhibitors has boosted interest in alternative targets. The discovery of several prostaglandin synthases and of distinct prostaglandin receptors has unraveled an unforeseen diversity within the prostanoid synthetic pathway. Behavioral and electrophysiological work in particular with genetically engineered mice meanwhile provides new clues to the role of different prostaglandins, prostaglandin synthases and prostaglandin receptors in pain pathways.

Keywords: Abbreviations; CFA; complete Freund's adjuvant; COX; cyclooxygenase (EC 1.14.99.1); CSF; cerebrospinal fluid; DRG; dorsal root ganglion; GlyRα3; glycine receptor α3 subunit; GST; glutathione; S; transferases; LPS; lipopolysaccharide; mPGES; microsomal prostaglandin E synthase (EC 5.3.99.3); NSAID; non-steroidal anti-inflammatory drug; PG; prostaglandin; PGIS; prostaglandin I synthase (EC 5.3.99.4); PKA; cAMP-dependent protein kinase; PKC; protein kinase C; PPAR; peroxisome proliferator activated receptor; RT-PCR; reverse transcriptase-polymerase chain reaction; TRPV1 channel; vanilloid type 1 transient receptor potential channel; TTX; tetrodotoxin; TXA2; thromboxane A2Pain; Nociception; Prostaglandin; Analgesia; Mouse mutant; NSAID

Prostanoids in nociception and pain by Hanns Ulrich Zeilhofer (pp. 165-174).

Accumulation of γ-globin mRNA and induction of irreversible erythroid differentiation after treatment of CML cell line K562 with new doxorubicin derivatives by Agata Szulawska; Justyna Arkusinska; Malgorzata Czyz (pp. 175-184).

Human chronic myelogenous leukemia (CML) cell line K562 can be chemically induced to differentiate and express embryonic and fetal globin genes. In this study, the effects of doxorubicin (DOX), an inducer of K562 cell erythroid differentiation, with those of epidoxorubicin (EDOX) as well as newly synthesized derivatives of both drugs (DOXM, DOXH, and EDOXM) on cell growth and differentiation were compared. Our results revealed that DOX, EDOX and their derivatives caused irreversible differentiation of K562 cells into more mature hemoglobin-containing cells. This phenomenon was linked to time-dependent inhibition of cell proliferation. Considering the impact of the structure of newly synthesized anthracyclines on their cellular activity, our data clearly indicated that among tested anthracyclines DOXM, a morpholine derivative of DOX exerted the highest antiproliferative and differentiating activity. An increase of γ-globin mRNA level caused both by high transcription rate and by mRNA stabilization, as well as an enhancement of expression but not activity of erythroid transcription factor GATA-1 were observed. Therefore, a high level of hemoglobin-containing cells in the presence of DOXM resulted from transcriptional and post-transcriptional events on γ-globin gene regulation. The same morpholine modification introduced to EDOX did not cause, however, similar effects on cellular level. Characterization of new powerful inducers of erythroid differentiation may contribute to the development of novel compounds for pharmacological approach by differentiation therapy to leukemia or to β-globin disorder, β-thalassemia.

Keywords: Abbreviations; ACLA; aclarubicin; CML; chronic myelogenous leukemia; DRB; daunorubicin; DOX; doxorubicin; EDOX; epidoxorubicin; DOXM; morpholine derivative of doxorubicin; EDOXM; morpholine derivative of epidoxorubicin; DOXH; hexamethyleneimine derivative of doxorubicinK562; Differentiation; Anthracycline derivatives; Erythroid gene expression; Growth arrest; mRNA stabilization

Rosiglitazone inhibits angiotensin II-induced CTGF expression in vascular smooth muscle cells––Role of PPAR-γ in vascular fibrosis by Deng-Feng Gao; Xiao-Lin Niu; Guang-Hua Hao; Ning Peng; Jin Wei; Ning Ning; Nan-Ping Wang (pp. 185-197).

Angiotensin (Ang) II plays a pivotal role in vascular fibrosis, which leads to serious complications in hypertension and diabetes. Connective tissue growth factor (CTGF) is a potent profibrotic factor implicated in the Ang II-induced pathologic fibrosis process. PPAR-γ activators thiazolidinediones have been recently reported to have beneficial vascular effects. However, their effects and related molecular mechanisms on extracellular matrix (ECM) turnover in vascular smooth muscle cells (VSMCs) are unknown. The present study evaluated the regulation of Ang II-induced CTGF, ECM production and cell growth by rosiglitazone in VSMCs. In aorta of Ang II-infused rats, CTGF expression was markedly increased, and type III collagen and fibronectin overexpression was observed. Cotreatment with rosiglitazone diminished these changes, whereas increased nuclear PPAR-γ expression in VSMCs. In growth-arrested VSMCs, rosiglitazone attenuated the proliferation and apoptosis, increased PPAR-γ production and activation, and reduced CTGF and ECM production in response to Ang II in a dose-dependent fashion. These inhibitory effects were attenuated by the pretreatment of cells with PPAR-γ antagonist GW9662 or bisphenol A diglycidyl ether (BADGE). Furthermore, rosiglitazone inhibited Ang II-induced Smad2 production and phosphorylation but had no effect on transforming growth factor-β₁ (TGF-β₁) expression. These results suggest that in Ang II-stimulated VSMCs, rosiglitazone caused an antiproliferative, antiapototic effect and reduces ECM production through mechanisms that include reducing CTGF expression, and a crosstalk between PPAR-γ and Smad may be involved in the inhibitory effects of rosiglitazone. This novel finding suggests a role of PPAR-γ activators in preventing Ang II-induced vascular fibrosis.

Keywords: Angiotensin; Connective tissue growth factor; Hypertension; PPAR-γ; Smooth muscle

Mechanism of action of AZD0865, a K⁺-competitive inhibitor of gastric H⁺,K⁺-ATPase by Karin Gedda; Carin Briving; Karin Svensson; Ingela Maxvall; Kjell Andersson (pp. 198-205).

AZD0865 is a member of a drug class that inhibits gastric H⁺,K⁺-ATPase by K⁺-competitive binding. The objective of these experiments was to characterize the mechanism of action, selectivity and inhibitory potency of AZD0865 in vitro. In porcine ion-leaky vesicles at pH 7.4, AZD0865 concentration-dependently inhibited K⁺-stimulated H⁺,K⁺-ATPase activity (IC₅₀ 1.0±0.2μM) but was more potent at pH 6.4 (IC₅₀ 0.13±0.01μM). The IC₅₀ values for a permanent cation analogue, AR-H070091, were 11±1.2μM at pH 7.4 and 16±1.8μM at pH 6.4. These results suggest that the protonated form of AZD0865 inhibits H⁺,K⁺-ATPase. In ion-tight vesicles, AZD0865 inhibited H⁺,K⁺-ATPase more potently (IC₅₀ 6.9±0.4nM) than in ion-leaky vesicles, suggesting a luminal site of action. AZD0865 inhibited acid formation in histamine- or dibutyryl-cAMP-stimulated rabbit gastric glands (IC₅₀ 0.28±0.01 and 0.26±0.003μM, respectively). In ion-leaky vesicles at pH 7.4, AZD0865 (3μM) immediately inhibited H⁺,K⁺-ATPase activity by 88±1%. Immediately after a 10-fold dilution H⁺,K⁺-ATPase inhibition was 41%, indicating reversible binding of AZD0865 to gastric H⁺,K⁺-ATPase. In contrast to omeprazole, AZD0865 inhibited H⁺,K⁺-ATPase activity in a K⁺-competitive manner ( K_i 46±3nM). AZD0865 inhibited the process of cation occlusion concentration-dependently (IC₅₀ 1.7±0.06μM). At 100μM, AZD0865 reduced porcine renal Na⁺,K⁺-ATPase activity by 9±2%, demonstrating a high selectivity for H⁺,K⁺-ATPase. Thus, AZD0865 potently, K⁺-competitively, and selectively inhibits gastric H⁺,K⁺-ATPase activity and acid formation in vitro, with a fast onset of effect.

Keywords: Abbreviations; PPI; proton pump inhibitor; DMSO; dimethylsulphoxide; MESG; 2-amino-6-mercapto-7-methyl-purine riboside; PNP; purine nucleoside phosphorylaseH; ⁺; ,K; ⁺; -ATPase/antagonists and inhibitors; Parietal cell; Gastric/drug effects; Selectivity; Mechanism of action; Fast onset

Mechanism of action of AZD0865, a K⁺-competitive inhibitor of gastric H⁺,K⁺-ATPase by Karin Gedda; Carin Briving; Karin Svensson; Ingela Maxvall; Kjell Andersson (pp. 198-205).

Role of tyrosine residues in modulation of claudin-4 by the C-terminal fragment of Clostridium perfringens enterotoxin by Motoki Harada; Masuo Kondoh; Chiaki Ebihara; Azusa Takahashi; Eriko Komiya; Makiko Fujii; Hiroyuki Mizuguchi; Shin-Ichi Tsunoda; Yasuhiko Horiguchi; Kiyohito Yagi; Yoshiteru Watanabe (pp. 206-214).

The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) modulates the barrier function of claudin-4 via its C-terminal 16 amino acids. In the current study, we investigated the roles of tyrosine residues (Y306, Y310 and Y312) in this region in the modulation of TJs by C-CPE. Single mutations of Y306, Y310 and Y312 to alanine resulted in partial reduction of claudin-4 binding. We also prepared double mutants of C-CPE to further evaluate the roles of these tyrosine residues. Replacement of Y310 and Y312 with alanine (Y310A/Y312A) partly reduced the ability of C-CPE to bind to claudin-4. Double mutants Y306A/Y310A and Y306A/Y312A, however, lost the ability to bind to claudin-4 and to modulate the TJ barrier. We also found that a triple mutant (Y306A/Y310A/Y312A) lost the ability to bind claudin-4, modulate the TJ barrier, and enhance jejunal absorption in rats. These results indicate that tyrosines 306, 310, and 312 are critical for the interaction of C-CPE with claudin-4 and for the modulation of TJ barrier function by C-CPE. This study provides information that should help in the development of claudin modulators based on C-CPE.

Keywords: Abbreviations; C-CPE; the C-terminal fragment of; Clostridium perfringens; enterotoxin; PSIF; protein synthesis inhibitory factor; TJ; tight junction; CPE; Clostridium perfringens; enterotoxin; TER; transepithelial electric resistance; C-CPE-PSIF; C-CPE fused to PSIF; PCR; polymerase chain reaction; LDH; lactate dehydrogenase; FD-4; fluorescein-isothiocyanate-dextran with a molecular weight of 4000Claudin-4; Clostridium perfringens; enterotoxin; Caco-2 cells; Tight junction; Barrier-function

Tangeretin suppresses IL-1β-induced cyclooxygenase (COX)-2 expression through inhibition of p38 MAPK, JNK, and AKT activation in human lung carcinoma cells by Kuan-Hung Chen; Meng-Shih Weng; Jen-Kun Lin (pp. 215-227).

Tangeretin (5,6,7,8,4′-pentamethoxyflavone) is a polymethoxylated flavonoid concentrated in the peel of citrus fruits. Recent studies have shown that tangeretin exhibits anti-proliferative, anti-invasive, anti-metastatic, and antioxidant activities. However, the anti-inflammatory properties of tangeretin are unclear. In this study, we examine the effects of tangeretin and its structure-related compound, nobiletin, on the expression of cyclooxygenases-2 (COX-2) in human lung epithelial carcinoma cells, A549, and human non-small cell lung carcinoma cells, H1299. Tangeretin exerts a much better inhibitory activity than nobiletin against IL-1β-induced production of COX-2 in A549 cells, and it effectively represses the constitutively expressed COX-2 in H1299. RT-PCR was used to investigate the transcriptional inhibition of COX-2 by tangeretin. COX-2 mRNA was rapidly induced by IL-1β in 3h and markedly suppressed by tangeretin. IL-1β-induced the activation of ERK, p38 MAPK, JNK, and AKT in A549 cells. COX-2 expression in response to IL-1β was attenuated by pretreatment with SB203580, SP600125, and LY294002, but not with PD98059, suggesting the involvement of p38 MAPK, JNK, and PI3K in this response. Pretreatment of cells with tangeretin inhibited IL-1β-induced p38 MAPK, JNK, and AKT phosphorylation and the downstream activation of NF-κB. These results may reveal that the tangeretin inhibition of IL-1β-induced COX-2 expression in A549 cells is, at least in part, mediated through suppression of NF-κB transcription factor as well as through suppression of the signaling proteins of p38 MAPK, JNK, and PI3K, but not of ERK.

Keywords: Tangeretin; COX-2; p38 MAPK; JNK; PI3K/AKT

Glutamate-stimulated peroxynitrite production in a brain-derived endothelial cell line is dependent on N-methyl-d-aspartate (NMDA) receptor activation by G.S. Scott; S.R. Bowman; T. Smith; R.J. Flower; C. Bolton (pp. 228-236).

There is accumulating and convincing evidence indicating a role for glutamate in the pathogenesis of the human demyelinating disease multiple sclerosis (MS). Studies in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, demonstrate that pharmacological inhibition of specific glutamate receptors suppresses neurological symptoms and prevents blood–brain barrier (BBB) breakdown. The mechanisms through which glutamate influences BBB function during EAE remain unclear. Glutamate triggers the production of nitric oxide and superoxide, which can lead to the formation of peroxynitrite (ONOO⁻). Recent studies have implicated ONOO⁻ in the loss of neurovascular integrity during EAE. We propose that glutamate contributes to BBB breakdown via the actions of ONOO⁻. The present investigation examined glutamate-induced ONOO⁻ formation in the b.End3 brain-derived endothelial cell line. b.End3 cells were incubated with a concentration range of glutamate and ONOO⁻ production was assessed over time. Results showed a concentration- and time-dependent increase in ONOO⁻ levels in glutamate-treated cells that were suppressed by selective and non-selective inhibitors of ONOO⁻-mediated reactions. Specific activation of b.End3-associated NMDA receptors also resulted in a concentration-dependent increase in ONOO⁻ production. The ability of b.End3 cells to respond to the presence of glutamate was confirmed through the detection of NMDA receptor immnuoreactivity in cell extracts. In addition, the use of the NMDA receptor antagonists MK-801 and memantine reduced glutamate-mediated ONOO⁻ generation from b.End3 cells. The data reinforce the important relationship between glutamate and the NMDA receptor, positioned at neurovascular sites, which may be of particular relevance to the pathogenesis of demyelinating disease.

Keywords: Peroxynitrite; Nitric oxide; Glutamate; N; -Methyl-; d; -aspartate receptor; Brain-derived endothelial cells

Melittin inhibits inflammatory target gene expression and mediator generation via interaction with IκB kinase by Hye Ji Park; Dong Ju Son; Chung Woo Lee; Myoung Suk Choi; Ung Soo Lee; Ho Sueb Song; Jeong Min Lee; Jin Tae Hong (pp. 237-247).

We previously found that bee venom (BV) and melittin (a major component of BV) has anti-inflammatory effect by reacting with the sulfhydryl group of p50 of NF-κB. Since the sulfhydryl group is present in IκB kinase (IKKα and IKKβ), anti-inflammatory effect of melittin via interaction with IKKs was investigated. We first examined binding of melittin to IKKs using surface plasmon resonance analyzer. Melittin binds to IKKα ( K_d=1.34×10⁻⁹M) and IKKβ ( K_d=1.01×10⁻⁹M). Consistent with the high binding affinity, melittin (5 and 10μg/ml) and BV (0.5, 1 and 5μg/ml) suppressed sodium nitroprusside, TNF-α and LPS induced-IKKβ and IKKβ activities, IκB release, and NF-κB activity as well as the expressions of iNOS and COX-2, and the generation of nitric oxide (NO) and prostaglandin E₂ (PGE₂) in Raw 264.7 mouse macrophages and synoviocytes obtained from rheumatoid arthritis patients. The binding affinities of melittin to mutant IKKs, was reduced, and the inhibitory effect of melittin on IKK and NF-κB activities, and NO and PGE₂ generation were abrogated by the reducing agents or in Raw 264.7 transfected with mutant plasmid IKKα (C178A) or IKKβ (C179A). These results suggest that melittin binding to the sulfhydryl group of IKKs resulted in reduced IKK activities, IκB release, NF-κB activity and generation of inflammatory mediators, indicating that IKKs may be also anti-inflammatory targets of BV.

Keywords: Abbreviations; BV; bee venom; COX; cyclooxygenase; cPLA; ₂; cytosolic phospholipase A; ₂; DTT; dithiothreitol; EMSA; electrophoretic mobility shift assay; GSH; glutathione; IκBs; inhibitors of κB; IKK; IκB kinase; iNOS; inducible nitric oxide synthase; LPS; lipopolysaccharide; NF-κB; nuclear factor-kappa B; NO; nitric oxide; PGs; prostaglandins; RA; rheumatoid arthritis; SNP; sodium nitroprusside; TNF-α; tumor necrosis factor-αBee venom; Melittin; NF-κB; IKK; Inflammation

Interaction of valerian extracts of different polarity with adenosine receptors: Identification of isovaltrate as an inverse agonist at A₁ receptors by Svenja K. Lacher; Ralf Mayer; Kathrin Sichardt; Karen Nieber; Christa E. Müller (pp. 248-258).

A series of extracts of valerian roots ( Valeriana officinalis L.) was prepared with solvents of different polarity. Polar as well as nonpolar extracts were found to interact with adenosine A₁ receptors. While polar extracts activated A₁ receptors (partial agonistic activity), nonpolar extracts showed antagonistic or inverse agonistic activity at A₁ receptors, as demonstrated by GTPγS binding assays at human recombinant A₁ receptors stably expressed in Chinese hamster ovary (CHO) cells. Guided by radioligand binding assays, fractionation of a lipophilic petroleum ether:diethyl ether (1:1) extract led to the isolation of isovaltrate, which was characterized as a potent, highly efficacious inverse agonist at adenosine A₁ receptors ( K_i rat A₁: 2.05μM). In experiments at rat brain slices measuring post-synaptic potentials (PSPs) in cortical neurons, isovaltrate at least partly reversed the reduction in the PSPs induced by the adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA). Isovaltrate may serve as a new lead structure for the development of inverse agonists at adenosine A₁ receptors. The common use of hydrophilic, but not lipophilic valerian extracts as mild sleep-inducing agents is consistent with the opposite actions of hydrophilic and lipophilic extracts on adenosine receptors.

Keywords: Abbreviations; CHO; Chinese hamster ovary; PSPs; post-synaptic potentials; ADA; adenosine deaminase; CCPA; 2-chloro-; N; ⁶; -cyclopentyladenosine; MSX-2; 3-(3-hydroxypropyl)-7-methyl-8-(; m; -methoxystyryl)-1-propargylxanthine; DTT; dithiothreitol; ACSF; artificial cerebrospinal fluid; CPA; N; ⁶; -cyclopentyladenosine; DPCPX; 8-cyclopentyl-1,3-dipropylxanthineValerian; Adenosine receptors; Bioassay-guided fractionation; Isovaltrate; A; ₁; antagonist; Inverse agonist

Molecular cloning, mutations and effects of NK₁ receptor antagonists reveal the human-like pharmacology of gerbil NK₁ receptors by Susanna Engberg; Ingela Ahlstedt; Agnes Leffler; Erik Lindström; Elin Kristensson; Arne Svensson; Ingrid Påhlman; Anders Johansson; Tomas Drmota; Bengt von Mentzer (pp. 259-269).

The present study investigates the pharmacology of the cloned neurokinin 1 receptor from the gerbil (gNK₁R), a species claimed to have human-like NK₁R (hNK₁R) pharmacology.The amino acid sequence of NK₁R was cloned. The hNK₁R, rat NK₁R (rNK₁R), gNK₁R and mutants of the gNK₁R were expressed in CHO cells. The affinity and potency of NKR agonists and the NK₁R antagonists CP99994 and RP67580 (NK₁R-selective) and ZD6021 (NK1/2R) were assessed in vitro by monitoring [³H]-SarMet SP binding and substance P-evoked mobilization of intracellular Ca²⁺. The gerbil foot tap (GFT) method was used to assess the potency of the antagonists in vivo.The gNK₁R coding sequence displayed an overall 95% and 97% homology with hNK₁R and rNK₁R, respectively. The affinity of the NK₁R-selective agonist³H-SarMet SP for human and gerbil NK₁R was similar (2.0 and 3.1nM) but lower for rNK₁R (12.4nM). The rank order potency of the agonists for NK₁R was SP≥ASMSP≥NKA⋙pro7NKB in all species. The NK₁R antagonists, ZD6021 and CP99994, had comparable affinity and potency for gerbil and human NK₁R, but were 1000-fold less potent for rNK₁R. In contrast, RP67580 had comparable affinity and potency for all three species. Mutations in positions 116 and 290 did not affect agonist potency at the gNK₁R while the potency of the antagonists ZD6021 and CP99994 were markedly decreased (10–20-fold). It is concluded that gNK₁R has similar antagonist pharmacology as the human-like orthologue and that species differences in antagonist function depend on key residues in the coding sequence and antagonist structure.

Keywords: Abbreviations; NK; neurokinin; SP; substance P; NKA; neurokinin A; Pro7NKB; [Pro; ⁷; ]-neurokinin B; ASMSP; acetyl-[Arg; ⁶; ,Sar; ⁹; ,Met(O; ₂; ); ¹¹; ]-SP6-11; NK; ₁; R; neurokinin 1 receptor; gNK; ₁; R; gerbil neurokinin 1 receptor; hNK; ₁; R; human neurokinin 1 receptor; rNK; ₁; R; rat neurokinin 1 receptor; mNK; ₁; R; mouse neurokinin 1 receptor; gpNK; ₁; R; guinea pig neurokinin 1 receptor; GFT; gerbil foot tap; Ile; isoleucine; CHO; Chinese Hamster Ovary; HPLC; high performance liquid chromatography; LC–MS; liquid chromatography–mass spectrometry; MRM; multiple reaction monitoringGerbil; Neurokinin 1 receptor cloning; Nueurokin receptor antagonists; Site directed mutagenesis; Gerbil foot tap; Homology models

Calmodulin potentiates Gβγ activation of phospholipase C-β3 by Jennifer S. McCullar; Dean A. Malencik; Walter K. Vogel; Kristi M. Crofoot; Sonia R. Anderson; Theresa M. Filtz (pp. 270-278).

Phospholipase C-β (PLC-β) isozymes (EC 3.1.4.11) hydrolyze the membrane phospholipid phosphatidylinositol-4,5-bisphosphate to generate intracellular second messenger signaling molecules inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) in response to receptor activation and other cellular stimuli. PLCβ1 and PLCβ3 isozymes were previously demonstrated to bind the calcium-sensitive molecule calmodulin [McCullar JS, Larsen SA, Millimaki RA, Filtz TM. Calmodulin is a phospholipase C-{beta} interacting protein. J Biol Chem 2003;278(36):33708–13]. We have now shown through fluorescence anisotropy that calmodulin/PLCβ3 affinities increase with increasing calcium in a physiologically relevant concentration range. The bimolecular affinity constants for calmodulin interaction with PLCβ1 or PLCβ3 were estimated as 260 and 200nM, respectively, from fluorescence anisotropy data. There was no effect of calmodulin on basal or Gαq-stimulated catalytic activity for either isozyme. However, the interaction between calmodulin and PLCβ3 leads to potentiation of activation by the G-protein βγ dimer in an in vitro assay. 1321N1 cells treated with calmodulin inhibitors concurrent with and post-stimulation of muscarinic receptors significantly reduced [3 H]PIP hydrolysis. Together these data are suggestive of cooperative role for calmodulin in the G-protein βγ dimer-stimulated activity of PLCβ3.

Keywords: Abbreviations; PLC; phosphatidylinositol phospholipid-specific phospholipase C; PE; phosphatidylethanolamine; PS; phosphatidylserine; PIP 16:0; synthetic phosphatidylinositol-4-phosphate with symmetric 16:0 saturated fatty acyl chains; PIP; phosphatidylinositol-4-phosphate; PIP2; phosphatidylinositol-4,5-bisphosphate; PIP3; phosphatidylinositol-3,4,5-trisphosphate; SDS-PAGE; sodium dodecyl phosphate polyacrylamide gel electrophoresis; W-13; N; -(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide; BSA; bovine serum albuminPhosphatidylinositol hydrolysis; Phospholipase C-beta; Gbetagamma; 1321N1 cells; Calmodulin; Fluorescence anisotropy

Calmodulin potentiates Gβγ activation of phospholipase C-β3 by Jennifer S. McCullar; Dean A. Malencik; Walter K. Vogel; Kristi M. Crofoot; Sonia R. Anderson; Theresa M. Filtz (pp. 270-278).

The effect of opiates on the activity of human placental aromatase/CYP19 by Olga L. Zharikova; Sujal V. Deshmukh; Meena Kumar; Ricardo Vargas; Tatiana N. Nanovskaya; Gary D.V. Hankins; Mahmoud S. Ahmed (pp. 279-286).

Aromatase, cytochrome P450 19, is a key enzyme in the biosynthesis of estrogens by the human placenta. It is also the major placental enzyme that metabolizes the opiatesl-acetylmethadol (LAAM), methadone, and buprenorphine (BUP). Methadone and BUP are used in treatment of the opiate addict and are competitive inhibitors of testosterone conversion to estradiol (E₂) and 16α-hydroxytestosterone (16-OHT) to estriol (E₃) by aromatase. The aim of this investigation is to determine the effect of 20 opiates, which can be administered to pregnant patients for therapeutic indications or abused, on E₂ and E₃ formation by placental aromatase. Data obtained indicated that the opiates increased, inhibited, or had no effect on aromatase activity. Their effect on E₃ formation was more pronounced than that on E₂ due to the lower affinity of 16-OHT than testosterone to aromatase. The K_i values for the opiates that inhibited E₃ formation were sufentanil, 7±1μM; LAAM, 13±8μM; fentanyl, 25±5μM; oxycodone, 92±22μM; codeine, 218±69μM; (+)-pentazocine, 225±73μM. The agonists morphine, heroin, hydromorphone, oxymorphone, hydrocodone, propoxyphene, meperidine, levorphanol, dextrorphan, and (−)-pentazocine and the antagonists naloxone and naltrexone caused an increase in E₃ formation by 124–160% of control but had no effect on E₂ formation. Moreover, oxycodone and codeine did not inhibit E₂ formation and the IC₅₀ values for fentanyl, sufentanil, and (+)-pentazocine were >1000μM. It is unlikely that the acute administration of the opiates that inhibit estrogen formation would affect maternal and/or neonatal outcome. However, the effects of abusing any of them during the entire pregnancy are unclear at this time.

Keywords: Abbreviations; 16-OHT; 16α-hydroxytestosterone; BSA; bovine serum albumin; BUP; buprenorphine; CYP19; cytochrome P450 19; E; ₂; 17β-estradiol; E; ₃; estriol; EDDP; 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine; LAAM; levo; -α-acetylmethadol; norBUP; norbuprenorphine; nor- and dinorLAAM; nor- and dinor-; levo; -α-acetylmethadolHuman placenta; Opiates; Aromatase/CYP19; Estrogen formation; Pregnancy

Identification of monoamine oxidase and cytochrome P450 isoenzymes involved in the deamination of phenethylamine-derived designer drugs (2C-series) by Denis S. Theobald; Hans H. Maurer (pp. 287-297).

In recent years, several compounds of the phenethylamine-type (2C-series) have entered the illicit drug market as designer drugs. In former studies, the qualitative metabolism of frequently abused 2Cs (2C-B, 2C-I, 2C-D, 2C-E, 2C-T-2, 2C-T-7) was studied using a rat model. Major phase I metabolic steps were deamination and O-demethylation. Deamination to the corresponding aldehyde was the reaction, which was observed for all studied compounds. Such reactions could in principal be catalyzed by two enzyme systems: monoamine oxidase (MAO) and cytochrome P450 (CYP). The aim of this study was to determine the human MAO and CYP isoenzymes involved in this major metabolic step and to measure the Michaelis–Menten kinetics of the deamination reactions. For these studies, cDNA-expressed CYPs and MAOs were used. The formation of the aldehyde metabolite was measured using GC–MS after extraction. For all compounds studied, MAO-A and MAO-B were the major enzymes involved in the deamination. For 2C-D, 2C-E, 2C-T-2 and 2C-T-7, CYP2D6 was also involved, but only to a very small extent. Because of the isoenzymes involved, the 2Cs are likely to be susceptible for drug–drug interactions with MAO inhibitors.

Keywords: Abbreviations; 2C-B; 4-bromo-2,5-dimethoxy-β-phenethylamine; 2C-I; 4-iodo-2,5-dimethoxy-β-phenethylamine; 2C-D; 2,5-dimethoxy-4-methyl-β-phenethylamine; 2C-E; 4-ethyl-2,5-dimethoxy-β-phenethylamine; 2C-T-2; 4-ethylthio-2,5-dimethoxy-β-phenethylamine; 2C-T-7; 2,5-dimethoxy-4-propylthio-β-phenethylamine; 5-HT; 5-hydroxy tryptamine (serotonin); MAO; monoamine oxidase; CYP; cytochrome P450; K; _m; Michaelis–Menten constant; V; _max; maximal turnover rate; PAR; peak area ratio; SIM; selected-ion monitoring; EI; electron ionization; IS; internal standard; LC–MS; liquid chromatography–mass spectrometry; APCI; atmospheric pressure chemical ionization; HPLC-UV; high performance liquid chromatography ultra violet detection2C-series; Designer drug; Metabolism; Cytochrome P450; Monoamine oxidase

Relevance of drug uptake and efflux for cisplatin sensitivity of tumor cells by Jochen Zisowsky; Susanne Koegel; Stefan Leyers; Krishna Devarakonda; Matthias U. Kassack; Maja Osmak; Ulrich Jaehde (pp. 298-307).

Platinum sensitivity and platinum resistance may involve altered activity of transport proteins. In order to assess the role of drug uptake and efflux in this phenomenon, we compared the expression of three copper transporters, intracellular platinum accumulation, DNA platination and cytotoxicity of cisplatin in two cisplatin-sensitive and -resistant tumor cell line pairs (ovarian A2780/A2780cis and cervical HeLa/HeLaCK cells). Gene expression of importer CTR1, and ATP7A and ATP7B efflux transporters (with and without cisplatin treatment) was investigated using quantitative real-time PCR and platinum concentrations were determined by flameless atomic absorption spectrometry.After incubation with cisplatin, DNA platination was significantly lower in the resistant variants compared to the respective sensitive cell lines, whereas no obvious difference in DNA repair was found. Accordingly, the resistant variants exhibited lower intracellular platinum concentrations than their respective parental cells (2.5- and 2.9-fold lower in A2780cis and HeLaCK cells, respectively). No differences in efflux were observed. Resistant cells expressed lower levels of CTR1 (1.5–1.8-fold) than their sensitive counterparts. Expression differences of ATP7A and ATP7B between resistant and sensitive cells were cell type-specific.The results highlight the relevance of CTR1 for cisplatin sensitivity as there is a clear relationship between lower CTR1 expression, intracellular concentration, DNA platination and cytotoxicity of cisplatin in both resistant cell lines. Our data provide the basis for a quantitative understanding of alterations in uptake and efflux processes leading to cisplatin resistance and might hence facilitate the development of ex vivo assays that can predict cisplatin sensitivity in tumor specimens of patients.

Keywords: Cisplatin; Accumulation; Uptake; Efflux; Resistance; TransportAbbreviations; ACTB; β-actin; ATP7A; ATPase, copper transporting, alpha polypeptide; ATP7B; ATPase, copper transporting, beta polypeptide; B2M; beta-2-microglobulin; hCTR1; human copper transporter 1; DEPC; diethyl pyrocarbonate; GSH; glutathione; GU; glucuronidase beta; HUPO; 60S (human) acidic ribosomal protein P1; HPRT1; hypoxanthine-phosphoribosyltransferase 1; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS; phosphate buffered saline; PhosA2; phospholipase A2; RPL13; ribosomal protein L13; TFR; transferrin receptor (p90; CD71); UBE2D2; ubiquitin-conjugating enzyme E2D2

Properties of flavonoids influencing the binding to bilitranslocase investigated by neural network modelling by Anna Karawajczyk; Viktor Drgan; Nevenka Medic; Ganiyu Oboh; Sabina Passamonti; Marjana Novi�? (pp. 308-320).

Bilitranslocase is a plasma membrane carrier firstly identified on the sinusoidal (vascular) domain of liver cells and later on also in the gastric epithelium. It transports diverse organic anions, such as bilirubin, some phthaleins and many dietary anthocyanins, suggesting that it could play a role both in the absorption of flavonoids from dietary sources and in their hepatic metabolism. This work was aimed at characterising the interaction of bilitranslocase with flavonols, a flavonoid sub-class. The results obtained show that, contrary to anthocyanins, flavonol glycosides do not interact with the carrier, whereas just some of the corresponding aglycones act as relatively poor ligands to bilitranslocase. These data point to a clear-cut discrimination between anthocyanins and flavonols occurring at the level of the bilitranslocase transport site. A quantitative structure–activity relationship based on counter propagation artificial neural network modelling was undertaken in order to shed light on the nature of flavonoid interaction with bilitranslocase. It was found that binding relies on the ability to establish hydrogen bonds, ruling out the involvement of charge interactions. This requisite might be at the basis of the discrimination between anthocyanins and flavonols by bilitranslocase and could lie behind some aspects of the distinct pharmacokinetic properties of anthocyanins and flavonols in mammals.

Keywords: Abbreviations; CP-ANN; counterpropagation artificial neural network; BSP; bromosulfophthaleinFlavonoids; Bilitranslocase; Transport; Liver; QSAR modelling; Counter propagation-artificial neural network

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Biochemical Pharmacology (v.73, #2)

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Biochemical Pharmacology (v.73, #2)