Check out our New Publishers' Select for Free Articles

Biochemical Pharmacology (v.69, #4)

Editorial Advisory Board (pp. iii).

Modulation of paraoxonase (PON1) activity by Lucio G. Costa; Annabella Vitalone; Toby B. Cole; Clement E. Furlong (pp. 541-550).

Paraoxonase 1 (PON1) is a serum enzyme closely associated with high density lipoprotein (HDL). PON1 hydrolyzes several organophosphorus compounds used as insecticides, as well as nerve agents; it metabolizes toxic oxidized lipids associated with both low density lipoprotein (LDL) and HDL; and it can hydrolyze a number of lactone-containing pharmaceutical compounds, inactivating some, while activating others. Serum PON1 activity in a given population can vary by 40-fold. Though most of this variation can be explained by polymorphisms in the coding region (Q192R) and the 5′ regulatory region (T-108C), modulation of PON1 by a variety of other factors should be taken into account, including other polymorphisms recently discovered but not yet characterized. This paper examines the major factors (environmental chemicals, drugs, smoking, alcohol, diet, age, disease conditions) that have been shown to modulate PON1 activity in either direction. As PON1 plays a protective role in organophosphate toxicity, and, because of its antioxidant capacity, in cardiovascular disease, a better understanding of how PON1 can be modulated by environmental factors has potential toxicological and clinical consequences.

Acetoxycycloheximide (E-73) rapidly induces apoptosis mediated by the release of cytochrome c via activation of c-Jun N-terminal kinase by Kimiko Kadohara; Yoshinori Tsukumo; Hikaru Sugimoto; Masayuki Igarashi; Kazuo Nagai; Takao Kataoka (pp. 551-560).

Cycloheximide (CHX) is an inhibitor of protein synthesis and commonly used to modulate death receptor-mediated apoptosis or to induce apoptosis in a number of normal and transformed cells. In this study we show that a close structural derivative of CHX, acetoxycycloheximide (E-73) induced rapid processing of procaspases and subsequent apoptosis with much higher efficacy than CHX in human leukemia Jurkat T cells. E-73 induced the release of cytochrome c from mitochondria even in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone. The Bcl-2 family protein Bcl-x_L suppressed cytochrome c release as well as processing of procaspases-3, -8, and -9 in E-73-treated cells. In Jurkat T cells transfected with the caspase-8 modulator FLIP_L, E-73 still induced activation of procaspase-3 and subsequent apoptosis, suggesting that the caspase-8 activity is dispensable for apoptosis. In contrast to CHX, E-73 drastically induced activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 MAP kinase. Inhibitory profiles of small-molecular kinase inhibitors revealed that JNK activation was critical for induction of cytochrome c release in E-73-induced apoptosis. Thus, our present results demonstrate that E-73, unlike CHX, induces strong activation of the JNK pathway and triggers rapid apoptosis mediated by the release of cytochrome c.

Keywords: Abbreviations; CHX; cycloheximide; ERK; extracellular signal-regulated kinase; FasL; Fas ligand; JNK; c-Jun N-terminal kinase; MCA; 4-methyl-coumaryl-7-amide; MTT; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; zVAD-fmk; benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluromethylketoneAcetoxycycloheximide; Apoptosis; c-Jun N-terminal kinase; Cycloheximide; Cytochrome; c; E-73

Transient up-regulation of P-glycoprotein reduces tacrolimus absorption after ischemia–reperfusion injury in rat ileum by Takanori Omae; Maki Goto; Masahiro Shimomura; Satohiro Masuda; Kimitaka Ito; Masahiro Okuda; Ken-ichi Inui (pp. 561-568).

Ischemia–reperfusion injury is an unavoidable problem for organ transplantation including small bowel transplantation, and causes a large intra-individual variation of tacrolimus (FK506) pharmacokinetics. Little information is available about the regulation of the intestinal P-glycoprotein expression during tissue regeneration. In the present study, we have examined the molecular and functional variations of ileum P-glycoprotein using rats after ischemia–reperfusion treatment. Morphological study revealed a rapid regeneration of the intestinal wall during 24h after reperfusion. A reverse transcription-coupled competitive PCR and Western blot analysis revealed that the intestinal expression of P-glycoprotein recovered with time after reperfusion. At 24h after reperfusion, the ileum P-glycoprotein level was transiently increased to two-fold, and the absorption rate of dihydro-[³H]FK506 from in situ ileum loop into portal vein was markedly low in comparison with the control. P-glycoprotein was detected in the crypt area as well as in villous cells at 6h after reperfusion, and then localized to the apical surface at 24h consistent with the cell proliferation and differentiation. However, the P-glycoprotein level returned to normal at 48h. The intra-individual variation in the absorptive rate of tacrolimus was suggested to be regulated by the morphological status of the intestinal epithelium and enterocyte expression level of P-glycoprotein. Therefore, the monitoring of the enterocyte P-glycoprotein level would provide useful information for determining the dosage of tacrolimus immediately after small bowl transplantation.

Keywords: Abbreviations; AUC; area under the concentration-time curve; BBM; brush-border membrane; C; /; D; ratio; concentration/oral dose ratio; CYP3A; cytochrome P450 IIIA; DAPI; 4′,6-diamidino-2-phenylindole; GAPDH; glyceraldehydes 3-phosphate dehydrogenase; MDR1; multidrug resistance; Pgp; P-glycoproteinMDR1; Small bowel transplantation; Proliferation; C219 monoclonal antibody; FK506; Transporter

Inhibition of tumor necrosis factor alpha secretion and prevention of liver injury in ethanol-fed rats by antisense oligonucleotides by Biddanda C. Ponnappa; Yedy Israel; Maria Aini; Feng Zhou; Rachel Russ; Qing-na Cao; Yiyang Hu; Raphael Rubin (pp. 569-577).

Elevated serum tumor necrosis factor alpha (TNF-α) levels predict mortality in patients with alcoholic liver disease. Administration of anti-TNF-α antibodies, obliteration of Kupffer cells or gut sterilization protect against ethanol-induced hepatocellular injury in animal models. In this study, we evaluated the in vivo efficacy of an antisense phosphorothioate oligodeoxynucleotide (S-ODN) targeted against TNF-α mRNA (TJU-2755). Naïve rats that were administered TJU-2755 (10mg/(kg body weight (BW)/day) for 2 days) in the free form were challenged with LPS to induce TNF-α secretion. Antisense TJU-2755 treatment reduced serum TNF-α levels by 62%. A comparison of the efficacies of mismatched and random S-ODNs with that of TJU-2755 showed that some non-specific inhibition might accompany the sequence-specific effects of TJU-2755. To optimize the targeting of the S-ODN, TJU-2755 was encapsulated in pH-sensitive liposomes for in vivo delivery to macrophages. The efficacy of liposome-encapsulated TJU-2755 was assessed in ethanol-fed animals that were administered LPS to induce liver injury. Liposomal delivery of TJU-2755 allowed a much lower dose (1.9mg/kgBW/day, for 2 days) of the S-ODN to reduce LPS-induced serum TNF-α (by 54%) and liver injury (by 60%) in ethanol-fed rats. These data indicate that liposome-encapsulated S-ODNs targeted against TNF-α have therapeutic potential in the treatment of alcoholic liver disease.

Keywords: Abbreviations; ALT; alanine aminotransferase; CHEMS; cholesteryl hemisuccinate; ICAM-1; inter cellular adhesion molecule-1; N.S.; not significant; PE; phosphatidylethanolamine; TE; Tris–EDTA; TNFR1; TNF-α receptor-1; T; _m; the melting temperature at which 50% of the oligonucleotide and its perfect hybrid are in duplex; S-ODN; phosphorothioate oligodeoxynucleotideAntisense; Liposomes; TNF-α; Ethanol; Lipopolysaccharide; Liver

Identification of key amino acids in the gastrin-releasing peptide receptor (GRPR) responsible for high affinity binding of gastrin-releasing peptide (GRP) by Tomoo Nakagawa; Simon J. Hocart; Michael Schumann; Jose A. Tapia; Samuel A. Mantey; David H. Coy; Kenji Tokita; Tatsuro Katsuno; Robert T. Jensen (pp. 579-593).

The bombesin (Bn) receptor family includes the gastrin-releasing peptide (GRPR) and neuromedin B (NMBR) receptors, Bn receptor subtype 3 (BRS-3) and Bn receptor subtype 4 (BB₄). They share 50% homology, yet their affinities for gastrin-releasing peptide (GRP) differ. The determinants of GRP high affinity for GRPR and BB₄, and low affinity for BRS-3 are largely unknown. To address this question we made an analysis of structural homologies in Bn receptor members correlated with their affinities for GRP to develop criteria to identify amino acids important for GRP selectivity. Fourteen differences were identified and each was mutated singly in GRPR to that found in hBRS-3. Eleven mutants had a loss of GRP affinity. Furthermore, three of four amino acids in the GRPR selected used a similar approach and previously reported to be important for high affinity Bn binding, were important for GRP affinity. Some GRPR mutants containing combinations of these mutations had greater decreases in GRP affinity than any single mutation. Particularly important for GRP selectivity were K101, Q121, A198, P199, S293, R288, T297 in GRPR. These results were confirmed by making the reverse mutations in BRS-3 to make GRP gain of affinity mutants. Modeling studies demonstrated a number of the important amino acids had side-chains oriented inward and within 6� of the binding pocket. These results demonstrated this approach could identify amino acids needed for GRP affinity and complemented results from chimera/mutagenesis studies by identifying which differences in the extracellular domains of Bn receptors were important for GRP affinity.

Keywords: Abbreviations; Bn; bombesin; BRS-3; bombesin receptor subtype 3; BB; ₄; bombesin receptor subtype 4; BSA; bovine serum albumin fraction V; DMEM; Dulbecco's minimum essential medium; DTT; dithiothreitol; EC; extracellular; FBS; fetal bovine serum; GRP; gastrin-releasing peptide; GRPR; gastrin-releasing peptide receptor; NMB; neuromedin B; NMBR; neuromedin B receptor; PBS; phosphate-buffered saline; TM; transmembraneGastrin-releasing peptide; Neuromedin B; Bombesin; Bombesin receptor; Gastrin-releasing peptide receptor; BRS-3

Inducible nitric oxide synthase-dependent stimulation of PKGI and phosphorylation of VASP in human embryonic kidney cells by Muriel Andr�; H�lia Latado; Emanuela Felley-Bosco (pp. 595-602).

Inducible nitric oxide synthase (iNOS) production of nitric oxide (NO) has been mostly associated with so-called nitrosative stress or interaction with superoxide anion. However, recent investigations have indicated that, as for the other isoenzymes producing NO, guanylyl cyclase (GC) is a very sensitive target of iNOS activity. To further investigate this less explored signaling, the NO-cyclic guanosine 3′–5′-monophosphate (NO–cGMP)-induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation on serine 239 was investigated in human embryonic kidney 293 cells (HEK cells). First, the expression and activity of α2 and β1 NO-sensitive GC subunits was determined by Western blot analysis, reverse transcription–polymerase chain reaction and NO donors administration. Then, the expression of a functional cGMP-dependent protein kinase I (PKGI) was verified by addition of 8-Br-cGMP followed by determination of phosphorylation of VASP on serine 239. Finally, iNOS activation of this signaling pathway was characterized after transfection of HEK cells with human iNOS cDNA. Altogether our data show that iNOS-derived NO activates endogenous NO-sensitive GC and leads to VASP phosphorylation in HEK cells.

Keywords: Abbreviations; cAMP; cyclic adenosine 3′–5′-monophosphate; cGMP; cyclic guanosine 3′–5′-monophosphate; DEA/NO; 2,2-diethyl-1-nitroso-oxyhydrasine; DETA/NO; (; Z; )-1-[(2-aminoethyl)-; N; -(2-ammonioethyl) amino] diazen-1-ium-1,2-diolate; GC; guanylyl cyclase; HEK cells; human embryonic kidney 293 cells; H89; N; -[2-(methylamino) ethyl]–5-isoquinolene-sulfonamide; IBMX; 3-isobutyl-1-methylxanthine; iNOS; inducible nitric oxide synthase; L-NMA; N; ^G; -monomethyl-; l; -arginine; NO; nitric oxide; ODQ; 1H-[1,2,4] oxadiazolo [4,3-a] quinoxylin-1-one; PDE; phosphodiesterase; PKGI; cGMP-dependent protein kinase I; PVP; polyvinylpyrrolidone; RT–PCR; reverse transcription–polymerase chain reaction; SNAP; S; -nitroso-; N; -acetyl-; d,l; -penicillamine; VASP; vasodilator-stimulated phosphoproteincGMP; NO donors; NO-sensitive guanylyl cyclase; PKGI; VASP; iNOS

Marker genes to predict sensitivity to FK228, a histone deacetylase inhibitor by Yuka Sasakawa; Yoshinori Naoe; Naoki Sogo; Takeshi Inoue; Tatsuya Sasakawa; Masahiko Matsuo; Toshitaka Manda; Seitaro Mutoh (pp. 603-616).

In this study, we detected genes sensitive to an histone deacetylase inhibitor, FK228 [( E)-(1 S,4 S,10 S,21 R)-7-[( Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, depsipeptide] in vitro and identified marker genes to predict sensitivity to FK228 in vivo using Affymetrix GeneChip. Three percent of genes (205/7070) were sensitive to FK228 in vitro, 105 and 100 genes, were up- and down-regulated, respectively, by FK228. Commonly up-regulated genes included p21^WAF1/Cip1, interleukin-8 (IL-8), histone family, JunB, caspase 9, mitogen-activated protein kinase phosphatase 1 (MKP-1) and mitogen-activated protein kinase (MAPK) family, and commonly down-regulated genes included cyclin A and MAPK family. One percent of genes (76/7070) showed native differences in patterns of expression, when FK228-sensitive (PC-3 prostate and SC-6-JCK (SC-6) stomach) and FK228-resistant (ACHN and A-498 renal) tumors implanted in BALB/c nu/ nu mice were compared. Twenty-seven and forty nine of those genes were expressed at high or low levels, respectively, in FK228-sensitive tumors. Caspase 9 and MKP-1 genes showed distinct differences in patterns of expression between FK228-sensitive and resistant tumors and have been known to have roles in apoptosis and chromatin remodeling. The expression of caspase 9 gene was higher in FK228-sensitive tumors and the expression of MKP-1 gene was higher in FK228-resistant tumors. Caspase 9 and MKP-1 genes in the other FK228-sensitive tumors had the same patterns of expression as they did in PC-3 and SC-6 tumors. Our results present profiles of gene expression related to FK228 and marker genes to predict sensitivity to FK228, such as caspase 9 and MKP-1 genes.

Keywords: Abbreviations; HDAC; histone deacetylase; TSA; trichostatin A; SAHA; suberoylanilide hydroxamic acid; SC-6; SC-6-JCK; LC-6; LC-6-JCK; nude; BALB/c; nu; /; nu; MKP-1; mitogen-activated protein kinase phosphatase 1; IL-8; interleukin-8; MAPK; mitogen-activated protein kinase; MKK; mitogen-activated protein kinase kinase; CML; chronic myeloid leukemia; JNK; Jun-N-terminal kinase; MAPKAPK; mitogen-activated protein kinase-activated protein kinase; ERK; extracellular signal-related kinaseFK228; FR901228; Histone deacetylase inhibitor; Caspase 9; MKP-1; GeneChip

Modulation of 5-fluorouracil cytotoxicity through thymidylate synthase and NF-κB down-regulation and its application on the radiolabelled iododeoxyuridine therapy on human hepatoma cell by Hsin-Ell Wang; Hui-Chuan Wu; Shang-Jyh Kao; Fan-Wei Tseng; Yu-Shan Wang; Hung-Man Yu; Shun-Lan Chou; Sang-Hue Yen; Kwan-Hwa Chi (pp. 617-626).

The inhibition of thymidylate synthase (TS) by 5-fluorouracil (5-FU) was known to increase the incorporation of radiolabelled iododeoxyuridine (IdUrd) into DNA. The relatively non-toxic compounds such as thiol-containing antioxidant pyrrolidinodithiocarbamte (PDTC) or aromatic fatty acid phenylbutyrate (PB) had been reported to enhance the cytotoxic efficacy of 5-FU. We designed a novel strategy through triplet combination of PB, PDTC and 5-FU to increase the radiolabelled IdUrd uptake and investigated the underlying mechanisms. The growth inhibition and [¹²⁵I]IdUrd-DNA incorporation by PB, PDTC, 5-FU in different combinations were tested on parent or p21^Waf1 transfected Hep3B cells. The combination of PB and PDTC was more effective in enhancing 5-FU cytotoxicity than either drug alone. The combination of PB/PDTC and 5-FU blocked cells in S-phase and resulted in 8.5-fold increase of radiolabelled IdUrd-DNA incorporation. The transfection of p21^Waf1 did not change the general pattern of enhancement. Intriguingly, the combination of PB and PDTC effectively down-regulated NF-κB and TS and prevented their up-regulation from 5-FU treatment than either drug alone through a p21^Waf1-independent mechanism. Based on this strategy, the 3-drug combination offered potential for improved radiolabelled IdUrd molecular radiotherapy for hepatoma treatment.

Keywords: Abbreviations; IdUrd; iododeoxyuridine; TS; thymidylate synthase; 5-FU; 5-fluorouracil; PDTC; pyrrolidinodithiocarbamte; PB; phenylbutyrate; PBS; phosphate buffered saline; DMEM; Dulbecco's modified Eagle's medium; dUMP; deoxyuridine monophosphate; dTMP; thymidine monophosphateIododeoxyuridine; Thymidylate synthase; 5-FU; PDTC; PB; NF-κB

Determination of the potency and subunit-selectivity of ribonucleotide reductase inhibitors with a recombinant-holoenzyme-based in vitro assay by Jimin Shao; Bingsen Zhou; Lijun Zhu; Angel J. Di Bilio; Leila Su; Yate-Ching Yuan; Shijun Ren; Eric J. Lien; Jennifer Shih; Yun Yen (pp. 627-634).

Ribonucleotide reductase (RR) is an important therapeutic target for anticancer drugs. The structure of human RR features a 1:1 complex of two homodimeric subunits, hRRM1 and hRRM2. p53R2 is a newly identified homologue of hRRM2. We have devised a holoenzyme-based in vitro assay for the determination of the potency and subunit-selectivity of small-molecule inhibitors of RR. The assay was implemented using two forms of recombinant RR (hRRM2/hRRM1 and p53R2/hRRM1) and based on their [³H]CDP reduction activity. Hydroxyurea was used to standardize the assay. We found that the activities of hRRM2/hRRM1 and p53R2/hRRM1 were decreased by hydroxyurea in a dose-dependent manner. The –NH–OH segment of hydroxyurea was shown to be essential for inhibition. In the presence of Fe(III) and reductants, less inhibition of enzymatic activity by hydroxyurea was observed, especially for p53R2/hRRM1. The potency of four hydroxyurea analogues (Schiff bases of hydroxysemicarbazide, SB-HSC) decreased in the order SB-HSC 21>SB-HSC 24>SB-HSC 2>hydroxyurea (HU)>SB-HSC 29. SB-HSC 2 and SB-HSC 24 inhibited p53R2/hRRM1 significantly more than hRRM2/hRRM1, whereas SB-HSC 21 and SB-HSC 29 showed low subunit-selectivity. Electron paramagnetic resonance (EPR) measurements showed that inhibition of RR was accompanied by reduction of its tyrosyl radical. The method was validated by comparison with data obtained using cell-based assays. We suggest that this novel recombinant-holoenzyme-based in vitro assay is a useful tool for the discovery of more potent and subunit-selective inhibitors of RR.

Keywords: Abbreviations; RR; ribonucleotide reductase; HU; hydroxyurea; SC; semicarbazide; SB-HSCs; Schiff bases of hydroxysemicarbazide; KB-; _WT; KB wild-type cells; KB-; _HUR; KB HU-resistant cells; EPR; electron paramagnetic resonance; QSAR; quantitative structure–activity relationship; DTT; dithiothreitolRibonucleotide reductase inhibitors; Potency and subunit-selectivity; In vitro assay; Two forms of holoenzyme; Recombinant subunit proteins

Effects of cannabinoids on nitric oxide production by chondrocytes and proteoglycan degradation in cartilage by Estery C. Mbvundula; Rowena A.D. Bunning; K.D. Rainsford (pp. 635-640).

Cannabinoids have been reported to have anti-inflammatory effects and reduce joint damage in animal models of arthritis. This suggests a potential therapeutic role in arthritis of this group of compounds. Cannabinoids were studied to determine whether they have direct effects on chondrocyte metabolism resulting in cartilage protection. Synthetic cannabinoids, R-(+)-Win-55,212 (Win-2) and S-(−)-Win-55,212 (Win-3) and the endocannabinoid, anandamide, were investigated on unstimulated or IL-1-stimulated nitric oxide (NO) production in bovine articular chondrocytes as well as on cartilage proteoglycan breakdown in bovine nasal cartilage explants. Win-2 significantly inhibited ( P<0.05) NO production in chondrocytes at 1–10μM concentrations. The combined CB₁ and CB₂ cannabinoid receptor antagonists, AM281 and AM630, respectively, at 100μM did not block this effect, but instead they potentiated it. Anandamide and Win-2 (5–50μM) also inhibited the release of sulphated glycosaminoglycans in bovine cartilage explants. The results suggest that some cannabinoids may prevent cartilage resorption, in part, by inhibiting cytokine-induced NO production by chondrocytes and also by inhibiting proteoglycan degradation.

Keywords: Abbreviations; AM281; 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-; N; -4-morpholinyl-1; H; -pyrazole-3-carboxamide; AM630; 6-iodo-2-methyl-1-[2-(4-morpholinyl) ethyl]-1; H; -indol-3-yl](4-methoxyphenyl)methanone; anandamide; N; -(2-hydroxyethyl)-5; Z; ,8; Z; ,11; Z; ,14; Z; -eicosatetraenamide; DMEM; Dulbecco's modified Eagle's medium; FBS; foetal bovine serum; IL-1α; interleukin-1 alpha; NO; nitric oxide; PBS; phosphate buffered saline; sGAG; sulphated glycosaminoglycan; Win-2; R; -(+)-Win-55,212 [; R; -(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate]; Win-3; S; -(−)-Win-55,212 [S-(−)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate]Cannabinoids; Cartilage resorption; Chondrocytes; Collagen; Interleukin-1α; Proteoglycan

Multiple P2X and P2Y receptor subtypes in mouse J774, spleen and peritoneal macrophages by Robson Coutinho-Silva; David M. Ojcius; Darek C. G�recki; Pedro M. Persechini; Rodrigo C. Bisaggio; Anderson N. Mendes; Joanne Marks; Geoffrey Burnstock; Philip M. Dunn (pp. 641-655).

We investigated P2 receptor expression and function in macrophages from mouse, and in the J774 cell line, and revealed a larger spectrum of P2 receptor subtypes than previously recognised. The nucleotides adenosine triphosphate (ATP), adenosine diphosphate, uridine triphosphate and uridine diphosphate evoked an increase in intracellular calcium and the activation of a potassium current. The sensitivity of these responses to the antagonists suramin, PPADS, MRS 2179 and Cibacron blue suggest the presence of at least three functional P2Y receptor subtypes, most probably P2Y₂, P2Y₄ and P2Y₆. ATP also activated P2X receptors, giving rise to a rapidly activating cation conductance. This response was insensitive to the antagonists suramin and Cibacron blue, was potentiated by Zn²⁺ and inhibited by acidification suggesting involvement of P2X₄ receptors. In low divalent cation solution, responses to ATP became larger, and dibenzoyl-ATP became more potent than ATP, indicating the presence of P2X₇ receptors. Immunofluorescence, flow cytometry, Western blots and RT-PCR show that P2X₄ and P2X₇ receptors are the most prominent in both macrophage types, while the expression of the other P2X subunits is variable and sometimes weak or undetectable. These techniques also demonstrated the presence of mRNA for P2Y₁, P2Y₂, P2Y₄ and P2Y₆ receptors along with protein expression for the three subtypes we investigated, namely, P2Y₁, P2Y₂ and P2Y₄.

Keywords: Abbreviations; BSS; balanced salt solution; NHS; normal horse serum; NGS; normal goat serum; MFI; mean fluorescence intensityAdenosine triphosphate; Purinergic receptors; Inflammation; Macrophages; Immunohistochemistry; Patch-clamp; Fura-2

Natural furocoumarins as inducers and inhibitors of cytochrome P450 1A1 in rat hepatocytes by Annette Baumgart; Melanie Schmidt; Hans-Joachim Schmitz; Dieter Schrenk (pp. 657-667).

Furocoumarins are natural plant constituents present in medicinal plants and in a variety of foods such as grapefruit juice. They are phototoxic and act as potent inhibitors of drug metabolism. We have investigated the interaction of four furocoumarins angelicin, bergamottin, isopimpinellin, and 8-methoxypsoralen with the expression and activity of aryl hydrocarbon receptor (AhR)-regulated CYP1A1 in rat hepatocytes in primary culture, both in the presence and absence of light. In intact hepatocytes pretreated with 2,3,7,8-tetrachlorodibenzo- p-dioxin and in microsomes isolated thereof, all furocoumarins tested acted as potent inhibitors of CYP1A1 activity bergamottin being the most potent inhibitor in microsomes with an IC₅₀ of 10nM in the presence and 60nM in the absence of light. 8-Methoxypsoralen and angelicin led to a significant induction of CYP1A1 mRNA in hepatocytes, while all furocoumarins except bergamottin increased xenobiotic-responsive element-driven reporter gene expression in transfected H4IIE rat hepatoma cells when light was excluded. Furthermore, all furocoumarins tested induced the expression of endogenous, immunoreactive CYP1A1 protein, primarily in the dark. In conclusion, our results demonstrate that individual furocoumarins present in food and medicinal plants can interfere with AhR-regulated CYP1A1 expression and activity in at least three major ways, i.e., (i) act as highly potent inhibitors of the catalytic activity of CYP1A1 both in the presence and absence of light, (ii) induce CYP1A1 gene expression in the absence of light via activation of the AhR, and (iii) induce CYP1A1 gene expression without activation of the AhR.

Keywords: Abbreviations; AhR; arylhydrocarbon receptor; ARNT; arylhydrocarbon receptor nuclear translocator; CYP; cytochrome P450; DMEM; Dulbecco's modified Eagle's medium; DMSO; dimethylsulfoxide; DTT; dithiothreitol; EROD; 7-ethoxyresorufin; O; -deethylase; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; LDH; lactate dehydrogenase; 5-MOP; 5-methoxypsoralen; 8-MOP; 8-methoxypsoralen; PAH; polycyclic aromatic hydrocarbons; PMSF; phenylmethylsulfonyl fluoride; PROD; 7-pentoxyresorufin; O; -depentylase; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxin; XRE; xenobiotic-responsive elementCYP1A1; Furocoumarins; Induction; Inhibition; Rat hepatocytes; TCDD

Assignment of voltage-gated potassium channel blocking activity to κ-KTx1.3, a non-toxic homologue of κ-hefutoxin-1, from Heterometrus spinifer venom by Selvanayagam Nirthanan; Joost Pil; Yousra Abdel-Mottaleb; Yuko Sugahara; Ponnampalam Gopalakrishnakone; Jeremiah S. Joseph; Kazuki Sato; Jan Tytgat (pp. 669-678).

A new family of weak K⁺ channel toxins (designated κ-KTx) with a novel “bi-helical�? scaffold has recently been characterized from Heterometrus fulvipes (Scorpionidae) venom. Based on the presence of the minimum functional dyad (Y5 and K19), κ-hefutoxin-1 (κ-KTx1.1) was investigated and found to block Kv 1.2 (IC₅₀ ∼40μM) and Kv 1.3 (IC₅₀ ∼150μM) channels. In the present study, κ-KTx1.3, that shares ∼60% identity with κ-hefutoxin 1, has been isolated from Heterometrus spinifer venom. Interestingly, despite the presence of the functional dyad (Y5 and K19), κ-KTx1.3 failed to reproduce the K⁺ channel blocking activity of κ-hefutoxin-1. Since the dyad lysine in κ-KTx1.3 was flanked by another lysine (K20), it was hypothesized that this additional positive charge could hinder the critical electrostatic interactions known to occur between the dyad lysine and the Kv 1 channel selectivity filter. Hence, mutants of κ-KTx1.3, substituting K20 with a neutral (K20A) or a negatively (K20E) or another positively (K20R) charged amino acid were synthesized. κ-KTx1.3 K20E, in congruence with κ-hefutoxin 1 with respect to subtype selectivity and affinity, produced blockade of Kv 1.2 (IC₅₀=36.8±4.9μM) and Kv 1.3 (IC₅₀=53.7±6.7μM) but not Kv 1.1 channels. κ-KTx1.3 K20A produced blockade of both Kv 1.2 (IC₅₀=36.9±4.9μM) and Kv 1.3 (IC₅₀=115.7±7.3μM) and in addition, acquired affinity for Kv 1.1 channels (IC₅₀=110.7±7.7μM). κ-KTx1.3 K20R failed to produce any blockade on the channel subtypes tested. These data suggest that the presence of an additional charged residue in a position adjacent to the dyad lysine impedes the functional block of Kv 1 channels produced by κ-KTx1.3.

Keywords: Abbreviation; K; ⁺; potassiumScorpion toxin; Voltage-gated potassium channel; Functional dyad; κ-Hefutoxin 1; κ-KTx1.3; Heterometrus spinifer

Neuronal nitric oxide modulates morphine antinociceptive tolerance by enhancing constitutive activity of the μ-opioid receptor by Erin L. Heinzen; Raymond G. Booth; Gary M. Pollack (pp. 679-688).

NO is a key mediator of morphine antinociceptive tolerance. This work was conducted to evaluate the specific effects of NO on μ-opioid receptor activity. To investigate the effects of morphine- andl-arginine (the NO precursor)-induced increases in NO, five groups of rats were treated with saline,l-arginine (100-, 300-, or 500-mg/kg/h), or morphine 3-mg/kg/h for 8h on Day 1; brain tissue was collected on Day 2. To evaluate the effects of additional increases in NO on morphine-induced alterations of the μ-opioid receptor, six groups of rats were treated with 8-h intravenous infusions for two consecutive days as per the following scheme (Day 1:Day 2): saline:saline (control); saline:morphine 3-mg/kg/h (tolerant);l-arginine 500-mg/kg/h:saline (NO control);l-arginine 100-mg/kg/h:morphine 3-mg/kg/h;l-arginine 300-mg/kg/h:morphine 3-mg/kg/h; andl-arginine 500-mg/kg/h:morphine 3-mg/kg/h (supertolerant). Brain tissue was collected at the end of Day 2. The time course of effects on morphine-induced receptor alterations due to increased NO also was evaluated. Brain tissue was analyzed for changes in radioligand (agonist and antagonist) binding and [³⁵S]GTPγS binding (agonist and antagonist). In the absence of agonist exposure, NO produced an alteration in the μ-opioid receptor that increased receptor activity. In the presence of agonist, NO increased constitutive activation of the μ-opioid receptor and reduced the ability of a selective μ-opioid agonist to activate the μ-opioid G-protein-coupled receptor; these molecular effects occurred in a time course consistent with the development of antinociceptive tolerance. This work establishes important NO-induced alterations in μ-opioid receptor functionality, which directly lead to the development of opioid antinociceptive tolerance.

Keywords: Abbreviations; DAMGO; [D-Ala; ²; , N-MePhe; ⁴; , Gly-ol; ⁵; ]enkephalin; GDP; guanosine diphosphate; GPCR; G-protein-coupled receptor; GppNHp; 5′guanylylimidodiphosphate; GTPγS; guanosine5′-G-(3-thio)triphosphate; NaCl; sodium chloride; NO; nitric oxideConstitutive activity; μ-Opioid receptor; Morphine; Opioid tolerance; Rats

Reductions in β-amyloid concentrations in vivo by the γ-secretase inhibitors BMS-289948 and BMS-299897 by Jeffery J. Anderson; Greg Holtz; Patricia P. Baskin; Mary Turner; Blake Rowe; Bowei Wang; Maria Z. Kounnas; Bruce T. Lamb; Donna Barten; Kevin Felsenstein; Ian McDonald; Kumar Srinivasan; Ben Munoz; Steven L. Wagner (pp. 689-698).

A primary pathological feature of Alzheimer's disease is β-amyloid (Aβ)-containing plaques in brain and cerebral vasculature. Reductions in the formation of Aβ peptides by γ-secretase inhibitors may be a viable therapy for reducing Aβ in Alzheimer's disease. Here we report on the effects of two orally active γ-secretase inhibitors. BMS-289948 (4-chloro- N-(2,5-difluorophenyl)- N-((1R)-{4-fluoro-2-[3-(1H-imidazol-1-yl)propyl]phenyl}ethyl)benzenesulfonamide hydrochloride) and BMS-299897 (4-[2-((1R)-1-{[(4-chlorophenyl)sulfonyl]-2,5-difluoroanilino}ethyl)-5-fluorophenyl]butanoic acid) markedly reduced both brain and plasma Aβ_1–40 in APP-YAC mice with ED₅₀ values of 86 and 22mg/kg per os (po), respectively, for BMS-289948, and 30 and 16mg/kg po, respectively, for BMS-299897. Both compounds also dose-dependently increased brain concentrations of APP carboxy-terminal fragments, consistent with inhibition of γ-secretase. BMS-289948 and BMS-299897 (100mg/kg po) reduced brain and plasma Aβ_1–40 rapidly (within 20min) and maximally within 3h. BMS-299897 also dose-dependently reduced cortical, cerebrospinal fluid (CSF), and plasma Aβ in guinea pigs with ED₅₀ values of 30mg/kg intraperitoneally, without affecting CSF levels of α-sAPP. The reductions in cortical Aβ correlated significantly with the reductions in both plasma ( r²=0.77) and CSF ( r²=0.61) Aβ. The decreases in Aβ were apparent at 3 and 6h post-administration of BMS-299897, but not at 12h. These results demonstrate that BMS-289948 and BMS-299897 are orally bioavailable, functional γ-secretase inhibitors with the ability to markedly reduce Aβ peptide concentrations in APP-YAC transgenic mice and in guinea pigs. These compounds may be useful pharmacologically for examining the effects of reductions in β-amyloid peptides in both animal models and in Alzheimer's disease.

Keywords: Abbreviations; Aβ; β-amyloid; APP; Amyloid precursor protein; DAPT; [; N; -(3,5-difluor-phenacetyl)-; l; -alanyl]-; S; -phenylglycine; BMS-289948; 4-chloro-; N; -(2,5-difluorophenyl)-; N; -((1R)-{4-fluoro-2-[3-(1H-imidazol-1-yl)propyl]phenyl}ethyl)benzenesulfonamide hydrochloride; BMS-299897; 4-[2-((1R)-1-{[(4-chlorophenyl)sulfonyl]-2,5-difluoroanilino}ethyl)-5-fluorophenyl]butanoic acid; CSF; cerebrospinal fluid; APP-YAC; amyloid precursor protein yeast artificial chromosome; ELISA; enzyme-linked immunosorbant assayAlzheimer's disease; β-Amyloid; Secretase; Amyloid precursor protein; γ-Secretase inhibitor; Proteolytic processing

Quantitative structure activity relationship studies on the flavonoid mediated inhibition of multidrug resistance proteins 1 and 2 by Jelmer J. van Zanden; Heleen M. Wortelboer; Sabina Bijlsma; Ans Punt; Mustafa Usta; Peter J. van Bladeren; Ivonne M.C.M. Rietjens; Nicole H.P. Cnubben (pp. 699-708).

In the present study, the effects of a large series of flavonoids on multidrug resistance proteins (MRPs) were studied in MRP1 and MRP2 transfected MDCKII cells. The results were used to define the structural requirements of flavonoids necessary for potent inhibition of MRP1- and MRP2-mediated calcein transport in a cellular model. Several of the methoxylated flavonoids are among the best MRP1 inhibitors (IC₅₀ values, ranging between 2.7 and 14.3μM) followed by robinetin, myricetin and quercetin (IC₅₀ values ranging between 13.6 and 21.8μM). Regarding inhibition of MRP2 activity especially robinetin and myricetin appeared to be good inhibitors (IC₅₀ values of 15.0 and 22.2μM, respectively). Kinetic characterization revealed that the two transporters differ marginally in the apparent K_m for the substrate calcein. For one flavonoid, robinetin, the kinetics of inhibition were studied in more detail and revealed competitive inhibition with respect to calcein, with apparent inhibition constants of 5.0μM for MRP1 and 8.5μM for MRP2. For inhibition of MRP1, a quantitative structure activity relationship (QSAR) was obtained that indicates three structural characteristics to be of major importance for MRP1 inhibition by flavonoids: the total number of methoxylated moieties, the total number of hydroxyl groups and the dihedral angle between the B- and C-ring. Regarding MRP2 mediated calcein efflux inhibition, only the presence of a flavonol B-ring pyrogallol group seems to be an important structural characteristic. Overall, this study provides insight in the structural characteristics involved in MRP inhibition and explores the differences between inhibitors of these two transporters, MRP1 and MRP2. Ultimately, MRP2 displays higher selectivity for flavonoid type inhibition than MRP1.

Keywords: Abbreviations; MRP; multidrug resistance protein; GSH; glutathione; GS-X; glutathione conjugate; IC; ₅₀; 50% inhibition concentration; P-gp; P-glycoprotein; QSAR; quantitative structure activity relationshipMRP1; MRP2; Flavonoids; QSAR; Calcein

Pretranslational upregulation of microsomal CYP4A in rat liver by intake of a high-sucrose, lipid-devoid diet containing orotic acid by Gloria M. Su; Eva Fiala-Beer; Jan Weber; Dennis Jahn; Graham R. Robertson; Michael Murray (pp. 709-717).

In rodents, high-fat diets promote hepatic lipid accumulation in rodents, activation of peroxisome proliferator activated receptor-α (PPARα) and upregulation of cytochrome P450 (CYP) 4A gene expression. Lipid-devoid diets containing sucrose and orotic acid (S/OA-diet) also cause lipid infiltration by stimulating intrahepatic lipid synthesis and preventing lipoprotein transport through the Golgi apparatus. This study evaluated the impact of the lipid-deficient S/OA-diet on CYP4A expression and PPARα activation in rodent liver. CYP4A protein and laurate ω-hydroxylation activity were increased in rat liver after S/OA-feeding for 21 days. CYP4A1 and CYP4A2 mRNAs were induced to 2.1- and 2.6-fold of control, but mRNAs corresponding to CYP4A3 and the peroxisomal acyl-CoA oxidase (AOX) were unchanged. Coadministration of clofibric acid and the S/OA-diet prevented hepatic lipid accumulation and upregulated CYP4A protein to levels comparable with clofibric acid alone (five-fold of control). Clofibric acid, alone and in combination with the S/OA-diet, upregulated CYP4A1-3 and AOX mRNAs. Hepatic PPARα protein was decreased by the S/OA-diet but was increased to 5.7-fold of control by clofibric acid; retinoid X-receptor-α (RXRα) protein was decreased to 26–41% of control by all treatments. In further studies, administration of the S/OA-diet to control and PPARα-null mice promoted hepatic lipid deposition; microsomal CYP4A protein was induced in wild-type but not PPARα-null mice. These findings implicate PPARα in the induction of CYP4A in rodent liver by the lipid-devoid S/OA-diet. Decreased availability of hepatic PPARα and RXRα after intake of the diet may contribute to the selective upregulation of hepatic CYP4A1 and CYP4A2 in this model.

Keywords: Abbreviations; AOX; acyl-CoA oxidase; CYP; cytochrome P450; S/OA-diet; sucrose/orotic acid-containing diet; PP; peroxisome proliferator; PPAR; peroxisome proliferator-activated receptor; RXR; retinoid X receptor; TBAR; thiobarbituric acid-reactive substanceCytochrome P450; Gene regulation; Orotic acid; Peroxisome proliferator-activated receptor-α; Retinoid X-receptor-α; Hepatic lipid synthesis

Sections

Personal tools

Biochemical Pharmacology (v.69, #4)

Sections

Personal tools

Local resources

Biochemical Pharmacology (v.69, #4)