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Biochemical Pharmacology (v.70, #7)

Editorial Advisory Board (pp. iii).

Non-COX-2 targets and cancer: Expanding the molecular target repertoire of chemoprevention by Khosrow Kashfi; Basil Rigas (pp. 969-986).

Chemoprevention represents a highly promising approach for the control of cancer. That nonsteroidal anti-inflammatory drugs (NSAIDs) prevent colon and other cancers has led to novel approaches to cancer prevention. The known inhibitory effect of NSAIDs on the eicosanoid pathway prompted mechanistic and drug development work focusing on cyclooxygenase (COX), culminating in clinical trials of cyclooxygenase 2 (COX-2) inhibitors for cancer prevention or treatment. However, two COX-2 inhibitors have been withdrawn due to side effects. Here we review several pathways of the eicosanoid cascade that are relevant to cancer; summarize the evidence regarding the role of COX-2 as a target for cancer prevention; and discuss several of the molecular targets that may mediate the chemopreventive effect of NSAIDs. The clinically modest results obtained to date with COX-2 specific inhibitors used in cancer prevention; the multiple COX-2-indpendent targets of both NSAIDs and COX-2 inhibitors; and the limitations of some COX-2 inhibitors indicate that exploiting these (non-COX-2) molecular targets will likely yield effective new approaches for cancer chemoprevention.

Keywords: COX-1; COX-2; COX-2 independent targets; Chemoprevention; Cancer; NSAIDs

Phosphorylation of 9-β-d-arabinofuranosylguanine monophosphate by Drosophila melanogaster guanylate kinase by Magnus Johansson; Marjan Amiri; Anna Karlsson (pp. 987-992).

Nucleoside monophosphate kinases have an important role in the synthesis of nucleotides that are required for cellular metabolism. These enzymes are also important for the phosphorylation of nucleoside- and nucleotide analogs used in cancer and anti-viral therapy. We report the cDNA cloning and characterization of a 23kDa guanylate kinase from Drosophila melanogaster ( Dm-GUK). The predicted amino acid sequence was 58% identical to the human guanylate kinase and the enzyme was shown to phosphorylate GMP and dGMP with ATP as phosphate donor. The monophosphates of the deoxyguanosine analogs 2′,2′-difluorodeoxyguanosine (dFdG) and 9-β-d-arabinofuranosylguanine (araG) were also shown to be phosphorylated by the enzyme. We used the enzyme to reconstitute the complete in vitro three-step phosphorylation pathway for the conversion of dGuo and araG to the corresponding triphosphates.

Keywords: Abbreviations; Dm; Drosophila melanogaster; dNK; deoxyribonucleoside kinase; GUK; guanylate kinase; araG; 9-β-; d; -arabinofuranosylguanine; dFdG; 2′,2′-difluorodeoxyguanosineNucleoside kinase; Nucleoside analog; Purine; Anti-cancer therapy; Nucleoside phosphorylation; Suicide gene therapy

NO-donating aspirin inhibits both the expression and catalytic activity of inducible nitric oxide synthase in HT-29 human colon cancer cells by Adam Spiegel; Thomas R. Hundley; Jie Chen; Jianjun Gao; Nengtai Ouyang; Xiaoping Liu; Mae F. Go; George J. Tsioulias; Khosrow Kashfi; Basil Rigas (pp. 993-1000).

Nitric oxide-releasing aspirin (NO-ASA) is emerging as a potentially important chemopreventive agent against colon cancer. We examined in HT-29 human colon adenocarcinoma cells the effect of NO-ASA on the inducible form of nitric oxide synthase (NOS2), an enzyme implicated in colon carcinogenesis. NO-ASA inhibited in a time- and concentration-dependent manner the expression of NOS2 up to 70% compared to control (IC₅₀ for this effect=46μM). NO-ASA also decreased the corresponding steady-state mRNA levels and this reduction preceded the reduction of protein levels by at least 6h. NO-ASA also reduced the enzymatic activity of NOS2, as determined by a direct enzyme assay (maximal reduction=80%) and by determining the accumulation of NO in the culture medium (IC₅₀ for this effect=36μM). These effects of NO-ASA on NOS2 were paralleled by inhibition in cell growth (IC₅₀=8.5μM). These findings indicate that NO-ASA profoundly inhibits both the expression and enzymatic activity of NOS2 and suggest that these effects may represent an important mechanism for the colon cancer chemopreventive effect of NO-ASA.

Keywords: Nitric oxide synthase; NOS2; Colon cancer; Aspirin; Nitric oxide-releasing aspirin; HT-29 human; Colon adenocarcinoma cells

Peptide inhibitors of G protein-coupled receptor kinases by Rainer Winstel; Hans-Georg Ihlenfeldt; G�nther Jung; Cornelius Krasel; Martin J. Lohse (pp. 1001-1008).

G protein-coupled receptor kinases (GRKs) are regulatory enzymes involved in the modulation of seven-transmembrane-helix receptors. In order to develop specific inhibitors for these kinases, we synthesized and investigated peptide inhibitors derived from the sequence of the first intracellular loop of the β₂-adrenergic receptor. Introduction of changes in the sequence and truncation of N- and C-terminal amino acids increased the inhibitory potency by a factor of 40. These inhibitors not only inhibited the prototypical GRK2 but also GRK3 and GRK5. In contrast there was no inhibition of protein kinase C and protein kinase A even at the highest concentration tested. The peptide with the sequence AKFERLQTVTNYFITSE inhibited GRK2 with an IC₅₀ of 0.6μM, GRK3 with 2.6μM and GRK5 with 1.6μM. The peptide inhibitors were non-competitive for receptor and ATP. These findings demonstrate that specific peptides can inhibit GRKs in the submicromolar range and suggest that a further decrease in size is possible without losing the inhibitory potency.

Keywords: Abbreviations; G; _βγ; βγ subunits of heterotrimeric GTP-binding proteins; GPCR; G protein-coupled receptor; GRK; G protein-coupled receptor kinase; IBMX; 3-isobutyl-1-methylxanthine; PKA; protein kinase A; PKC; protein kinase C; ROS; rod outer segmentsG-protein-coupled receptor kinases; Receptor desensitization; Phosphorylation; Non-competitive inhibition

A high-affinity monoclonal antibody with functional activity against the 5-hydroxytryptaminergic (5-HT₄) receptor by Rehab Kamel; Pierre Eftekhari; Simone Garcia; Magali Berthouze; Isabelle Berque-Bestel; Jean-Christophe Peter; Frank Lezoualc’h; Johan Hoebeke (pp. 1009-1018).

Splenocytes from a BALB/c mouse immunised with a synthetic peptide corresponding to the second extracellular loop of the 5-HT₄ receptor were fused with SP2/O myeloma cells to produce a monoclonal antibody. The monoclonal antibody was of the IgG2b isotype. The antibody recognised the human 5-HT_4(g) (h5-HT_4(g)) receptor by immunoblots and by immunofluorescence on chinese hamster ovary (CHO) cells expressing this 5-HT₄ receptor isoform. Epitope mapping of the antibody suggested the recognition of a conformational epitope, encompassing the N- and C-terminal fragments of the second extracellular loop. Kinetic experiments using surface plasmon resonance showed that the antibody had a picomolar affinity for its cognate peptide. Inhibition experiments using the same methodology confirmed the specificity of the interaction. The antibody at a concentration of 500pM competitively inhibited inverse agonist GR113808 binding and showed an inverse agonist effect on the basal activity of CHO cells expressing the 5-HT_4(g) receptor. The antibody decreased the effect of 5-HT at 500 and 50pM concentrations but it increased 5-HT-induced cAMP levels at 5pM. The dual effect of the monoclonal antibody could be ascribed to mono- or bivalent recognition of the receptor. The antibody described here is the first example of a high-affinity modulator of the 5-HT₄ receptor.

Keywords: Abbreviations; CHO cells; Chinese hamster ovary cells; GPCR; G protein-coupled receptors; RU; resonance units; SDS-PAGE; sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SPR; surface plasmon resonance5-HT; ₄; receptor; Antagonist; cAMP; Epitope mapping; Monoclonal antibody; Surface plasmon resonance

Cloning, high level expression of human paraoxonase-3 in Sf9 cells and pharmacological characterization of its product by Haiqin Lu; Jie Zhu; Yuhui Zang; Yuguan Ze; Junchuan Qin (pp. 1019-1025).

Human paraoxnase-3 (hPON3) (EC3.1.8.1) is a lipid-associated enzyme with antioxidant activity, and can inhibit the oxidation of low-density lipoprotein (LDL), thereby inhibiting early atherogenic process. In the present study, human PON3 gene was cloned from Human Fetal Liver Marathon-Ready cDNA and expressed in insect cells using baculovirus vector. Twenty-eight milligrams of purified recombinant hPON3 (rhPON3) was obtained from 1L Sf9 cells culture. The K_m and V_max values of rhPON3, with respective to phenylacetate hydrolysis were 7.46�4.40mM and 89�10.54U/mg ( n=3). The kinetic parameters of V_max and K_m for dihydrocoumarin hydrolysis by rhPON3 were 698�248U/mg and 0.84�0.24mM ( n=3). LDL oxidation assay indicated that rhPON3 could effectively protect LDL against copper-induced oxidation in vitro.

Keywords: Human paraoxonase-3; Baculovirus-mediated expression system; Sf9 cell; Gene expression; LDL oxidation; Dihydrocoumarin

N-Acetyl-l-cysteine suppresses TGF-β signaling at distinct molecular steps: The biochemical and biological efficacy of a multifunctional, antifibrotic drug by Steffen K. Meurer; Birgit Lahme; Lidia Tihaa; Ralf Weiskirchen; Axel M. Gressner (pp. 1026-1034).

The interrelated signaling via TGF-β1 and reactive oxygen species has a profound impact on fibrogenesis and is therefore selected as target for antifibrotic therapies. This prompted us to investigate the influence of the antioxidant N-acetyl-l-cysteine on TGF-β signaling in culture-activated hepatic stellate cells, the most relevant pro-fibrogenic cell type in liver. Dissection of the molecular steps involved in TGF-β signaling revealed that N-acetyl-l-cysteine dose-dependently abrogated the induction of the TGF-β1 signaling reporter gene activation, the phosphorylation of Smad2 and Smad3, and the up-regulation of Smad7 mRNA. By means of Western blot analysis and cross-linking experiments, it was demonstrated that these effects are based on disintegration of TGF-β1 and the TGF-β receptor endoglin, as well as a reduced ligand binding capacity of betaglycan. We conclude that N-acetyl-l-cysteine is a specific inhibitor of TGF-β signaling targeting different components of the TGF-β signaling machinery. In conclusion, these findings suggest that this non-toxic aminothiol downregulates TGF-β signal transduction thereby mediating beneficial effects on experimental liver fibrosis characterized by TGF-β hyperactivity.

Keywords: Abbreviations; Ad-CA-ALK-5; adenovirus expressing a constitutively active TGF-β receptor type I; BSO; l; -buthionine-; S; ,; R; -sulfoximine; DTT; dithiothreitol; FCS; fetal calf serum; HSC; hepatic stellate cell(s); Luc; luciferase; MLP; major late promoter; MOI; multiplicity of infection; NAC; N; -acetyl-; l; -cysteine; ROS; reactive oxygen species; TGF-β; transforming growth factor β; TβR; TGF-β receptorFibrosis; Liver; Reactive oxygen; Receptor signaling; Smad

Bile acid-induced proliferation of a human colon cancer cell line is mediated by transactivation of epidermal growth factor receptors by Kunrong Cheng; Jean-Pierre Raufman (pp. 1035-1047).

Although epidemiological studies indicate an association between elevations in fecal bile acids and the development of colorectal cancer, the cellular mechanism for the proliferative actions of bile acids is not clear. Studies from other laboratories indicate a paradoxical pro-apoptotic action of bile acids on cell culture lines. Our previous studies indicate that cholinergic agonist-induced proliferation of colon cancer cells that express M₃ muscarinic receptors (M₃R) is mediated by transactivation of the epidermal growth factor receptor (EGFR) and that bile acids stimulate proliferation of colon cancer cells that express M₃R. In the present study, we investigated the effects of bile acids on cell signaling and proliferation of a human colon cancer cell line (H508 cells) that abundantly expresses M₃R and EGFR. Treatment with taurine and glycine conjugates of lithocholic and deoxycholic acids stimulated reversible activation of the p44/42 MAP kinase signaling cascade and proliferation of H508 cells. Bile acids did not stimulate proliferation of SNU-C4 colon cancer cells that express EGFR but not muscarinic receptors. Atropine, a muscarinic receptor inverse agonist, blocked bile acid-induced H508 cell proliferation. At concentrations that stimulate cell proliferation, conjugated bile acids did not activate caspase-3, a key mediator of apoptosis. Conjugated bile acids stimulated phosphorylation of EGFR Tyr992, thereby implicating EGFR transactivation in the cellular mechanism underlying their proliferative actions. This was confirmed by observing that inhibitors of EGFR activation and antibodies to the ligand-binding domain of EGFR blocked both the signaling and proliferative actions of bile acids. Collectively, these results suggest that in this colon cancer cell line, bile acid-induced colon cancer cell proliferation is M₃R-dependent and is mediated by transactivation of EGFR.

Keywords: Abbreviations; MAPK; mitogen activated protein kinase; M; ₃; R; M; ₃; subtype muscarinic receptor; ACh; acetylcholine; p90RSK; p90 ribosomal S kinase; EGFR; epidermal growth factor receptor; DCT; deoxycholyltaurine; DCG; deoxycholylglycine; LCT; lithocholyltaurine; GPCR; G-protein coupled receptor; RTK; receptor tyrosine kinaseBile acids; Colon cancer; Epidermal growth factor receptor; Muscarinic receptors; Acetylcholine; Signal transduction; Proliferation

Resveratrol inhibits type II phosphatidylinositol 4-kinase: A key component in pathways of phosphoinositide turn over by Rupa Srivastava; Aparna Ratheesh; Rajiv K. Gude; K.V.K. Rao; Dulal Panda; Gosukonda Subrahmanyam (pp. 1048-1055).

Resveratrol has anti-inflammatory, cardio protective and cancer chemopreventive properties. The molecular targets for resveratrol in early signaling cascades are not well understood. Resveratrol inhibits type II PtdIns 4-kinase but not PtdIns 3-kinase activity in vitro. Resveratrol directly binds to the enzyme with a K_d of 7.2μM. Kinetic studies show that resveratrol competes with PtdIns binding. Inhibition of PtdIns 4-kinase activity by resveratrol/phenylarsine oxide reduces Jurkat cell adhesion to matrigel/fibronectin coated surfaces, suggesting a role for type II PtdIns 4-kinase in lymphocyte infiltration to the sites of inflammation.

Keywords: Abbreviations; PtdIns (4,5); P; ₂; d; -; myo; -phosphatidylinositol 4,5-bisphosphate; PtdIns 4; P; d; -; myo; -phosphatidylinositol 4-phosphate; PtdIns; d; -; myo; -phosphatidylinositolSignal transduction; T lymphocytes; Cell adhesion

MRP1 mutated in the L₀ region transports SN-38 but not leukotriene C₄ or estradiol-17 (β-d-glucuronate) by Tomohiro Noguchi; Xiao-Qin Ren; Shunji Aoki; Yoshinobu Igarashi; Xiao-Fang Che; Yuichi Nakajima; Homare Takahashi; Ryoichi Mitsuo; Kazutake Tsujikawa; Tomoyuki Sumizawa; Misako Haraguchi; Motomasa Kobayashi; Susumu Goto; Minoru Kanehisa; Takashi Aikou; Shin-ichi Akiyama; Tatsuhiko Furukawa (pp. 1056-1065).

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that confers multidrug resistance on tumor cells. Much convincing evidence has accumulated that MRP1 transports most substances in a GSH-dependent manner. On the other hand, several reports have revealed that MRP1 can transport some substrates independently of GSH; however, the importance of GSH-independent transport activity is not well established and the mechanistic differences between GSH-dependent and -independent transport by MRP1 are unclear.We previously demonstrated that the amino acids W₂₆₁ and K₂₆₇ in the L₀ region of MRP1 were important for leukotriene C₄ (LTC₄) transport activity of MRP1 and for GSH-dependent photolabeling of MRP1 with azidophenyl agosterol-A (azidoAG-A). In this paper, we further tested the effect of W222L, W223L and R230A mutations in MRP1, designated dmL₀MRP1, on MRP1 transport activity. SN-38 is an active metabolic form of CPT-11 that is one of the most promising anti-cancer drugs. Membrane vesicles prepared from cells expressing dmL₀MRP1 could transport SN-38, but not LTC₄ or estradiol-17 (β-d-glucuronate), and could not be photolabeled with azidoAG-A. These data suggested that SN-38 was transported by a different mechanism than that of GSH-dependent transport. Understanding the GSH-independent transport mechanism of MRP1, and identification of drugs that are transported by this mechanism, will be critical for combating MRP1-mediated drug resistance. We performed a pairwise comparison of compounds that are transported by MRP1 in a GSH-dependent or -independent manner. These data indicated that it may be possible to predict compounds that are transported by MRP1 in a GSH-independent manner.

Keywords: Abbreviations; ABC transporter; ATP-binding cassette transporter; ACNU; 3-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-1-(2-chloroethyl)-1-nitrosourea; ADM; adriamycin; AG-A; agosterol A; azidoAG-A; [; ¹²⁵; I] 11-azidophenyl agosterol A; AZT; azidothymidine; BSO; buthionine sulfoximine; BSP; sulfobromophthalein; C; COOH; CDDP; cis; -platinum(II) diammine dichloride; d4T; 2′,3′-didehydro-3′-deoxythymidine; DAPI; 4′,6-diamidino-2-phenylindole dihydrochloride; DIDS; 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; DTT; dithiothreitol; E; ₂; 17β-G; estradiol-17 (β-; d; -glucuronate); γGCS; γ-glutamylcysteine synthetase; IAARh123; iodoaryl azidorhodamine 123; IAAQ; N; -{4-[1-hydroxy-2-(dibutylamino) ethyl] quinolin-8-yl}-4-azidosalicylamide; IACI; N; -(hydrocinchonidin-8′-yl)-4-azido-2-hydroxybenzamide; LTC; ₄; leukotriene C; ₄; LTD; ₄; leukotriene D; ₄; LTE; ₄; leukotriene E; ₄; mAMSA; 4′-(9-acridinylamino)methanesulfon-; m; -anisidide; MDR; multidrug resistance; MRP1; multidrug resistance protein 1; MV; membrane vesicles; N; NH; ₂; NBD; nucleotide-binding domain; NNAL-; o; -glucuronide; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol-; o; -glucuronide; P-gp; P-glycoprotein; PMEA; 9-(2-phosphonylmethoxyethyl) adenine; PMEG; 9-(2-phosphonylmethoxyethyl) guanine; 3TC; 2′,3′-dideoxy-3′-thiacytidine; TM; transmembrane segment; TMD; transmembrane domain; VCR; vincristine; VP-16; etoposide; Rh123; rhodamine 123MRP1; SN-38; GSH; L; ₀; ABC transporter; Drug resistance; LTC; ₄; E; ₂; 17β-G

Inhibitory effects of rosmarinic acid on adriamycin-induced apoptosis in H9c2 cardiac muscle cells by inhibiting reactive oxygen species and the activations of c-Jun N-terminal kinase and extracellular signal-regulated kinase by Do-Sung Kim; Hyung-Ryong Kim; Eun-Rhan Woo; Seong-Tshool Hong; Han-Jung Chae; Soo-Wan Chae (pp. 1066-1078).

Rosmarinic acid (RA) is a naturally occurring polyphenolic and is found in several herbs in the Lamiaceae family, such as, Perilla frutescens. ADR is a potent anti-tumor drug, but is unfortunately potently cardiotoxic. This study was undertaken to investigate the inhibitory effect of RA on ADR-induced apoptosis in H9c2 cardiac muscle cells at a mechanistic level. In vitro, ADR significantly decreased the viabilities of H9c2 cells, and this was accompanied by apoptotic features, such as a change in nuclear morphology and caspase protease activation. RA was found to markedly inhibit these apoptotic characteristics by reducing intracellular ROS generation and by recovering the mitochondria membrane potential (Δ ψ). In addition, RA reversed the downregulations of GSH, SOD and Bcl-2 by ADR. In the present study, ADR was found to activate c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), transcriptional factor-activator-protein (AP)-1. We found that c-fos, Jun-B, Jun-D and p-c-Jun were super shifted by ADR, indicating that these proteins have an important role in the ADR-induced AP-1 activation. The inhibitions of JNK and ERK using appropriate inhibitors or dominant negative cell lines reduced ADR-induced apoptosis in H9c2 cardiac muscle cells. Taken together, these results suggest that RA can inhibit ADR-induced apoptosis in H9C2 cardiac muscle cells by inhibiting ROS generation and JNK and ERK activation. Thus, we propose that RA should be viewed as a potential chemotherapeutic that inhibits cardiotoxicity in ADR-exposed patients.

Keywords: Abbreviations; ADR; ADR; AP-1; activator protein-1; BCA; bicinchoninic acid; CAT; catalase; DiOC; ₆; (3); 3,3′-diexyloxocarbocyanine iodide; DMEM; Dulbecco's modified Eagle's medium; DMSO; dimethyl sulfoxide; DN; dominant negative; EMSA; electrophoretic mobility shift assay; ERK; extracellular signal-regulated kinase; FBS; fetal bovine serum; GSH; glutathione; JNK; c-Jun N-terminal Kinase; MAN; mannitol; MAPK; mitogen-activated protein kinase; NAC; N; -acetyl-cysteine; RA; rosmarinic acid; ROS; reactive oxygen species; SOD; superoxide dismutaseRosmarinic acid; ROS; Adriamycin; JNK; ERK; AP-1; Apoptosis

IRAS, a candidate for I₁-imidazoline receptor, mediates inhibitory effect of agmatine on cellular morphine dependence by Ning Wu; Rui-Bin Su; Bo Xu; Xin-Qiang Lu; Yin Liu; Jian-Quan Zheng; John E Piletz; Jin Li; Bo-Yi Qin (pp. 1079-1087).

Agmatine, an endogenous ligand for the I₁-imidazoline receptor, has previously been shown to prevent morphine dependence in rats and mice. To investigate the role of imidazoline receptor antisera-selected protein (IRAS), a strong candidate for I₁R, in morphine dependence, two CHO cell lines were created, in which μ opioid receptor (MOR) was stably expressed alone (CHO-μ) or MOR and IRAS were stably co-expressed (CHO-μ/IRAS). After 48h administration of morphine (10μM), naloxone induced a cAMP overshoot in both cell lines, suggesting cellular morphine dependence had been produced. Agmatine (0.1–2.5μM) concentration-dependently inhibited the naloxone-precipitated cAMP overshoot when co-pretreated with morphine in CHO-μ/IRAS, but not in CHO-μ. Agmatine at 5–100μM also inhibited the cAMP overshoot in CHO/μ and CHO-μ/IRAS. Efaroxan, an I₁R-preferential antagonist, completely blocked the effect of agmatine on the cAMP overshoot at 0.1–2.5μM in CHO-μ/IRAS, while partially reversing the effects of agmatine at 5–100μM.l-type calcium channel blocker nifedipine entirely mimicked the effects of agmatine at high concentrations on forskolin-stimulated cAMP formation in CHO-μ and naloxone-precipitated cAMP overshoot in morphine-pretreated CHO-μ. Therefore, IRAS, in the co-transfected CHO-μ/IRAS cell line, appears necessary for low concentrations of agmatine to cause attenuation of cellular morphine dependence. An additional effect of agmatine at higher concentrations seems to relate to both transfected IRAS and some naive elements in CHO cells, andl-type voltage-gated calcium channels are not ruled out. This study suggests that IRAS mediates agmatine's high affinity effects on cellular morphine dependence and may play a role in opioid dependence.

Keywords: Abbreviations; α; ₂; -AR; α; ₂; -adrenoceptors; Agm; agmatine; APH; aminoglycoside phosphotransferases; BU224; 2-(4,5-dihydroimidaz-2-yl)-isoquinolin; CHO; Chinese hamster ovary; CHO-μ; Chinese hamster ovary cells expressing μ opioid receptor; CHO-μ/IRAS; Chinese hamster ovary cells co-expressing μ opioid receptor and imidazoline receptor antisera-selected; CHO-μ/IRAS-Low; Chinese hamster ovary cells co-expressing μ opioid receptor and low level imidazoline receptor antisera-selected; Efa; efaroxan; GABA; γ-amino butyric acid; hIRAS; human imidazoline receptor antisera-selected protein; IBMX; 3-isobutyl-1-methylxanthine; I; ₁; R; I; ₁; -imidazoline receptor; I; ₂; R; I; ₂; -imidazoline receptor; IRAS; imidazoline receptor antisera-selected protein; Irs; imidazoline receptors; l; -NAME; N; ω-nitro-; l; -arginine; MK801; dizocilpine; Mor; morphine; MOR; μ opioid receptor; NE; norepinephrine; Nif; nifedipine; NMDA; N; -methyl-; d; -aspartate; NOS; nitric oxide synthase; rMOR; rat μ opioid receptor; VTA; ventral tegmental areaOpioid dependence; Imidazoline receptor; IRAS; Agmatine; cAMP overshoot

Up-regulation of Mrp4 expression in kidney of Mrp2-deficient TR⁻ rats by Chuan Chen; Angela L. Slitt; Mathew Z. Dieter; Yuji Tanaka; George L. Scheffer; Curtis D. Klaassen (pp. 1088-1095).

Multidrug resistance-associated proteins (Mrps) are a group of ATP-dependent efflux transporters for organic anions. Mrp2 and Mrp4 are co-localized to the apical (brush-border) membrane domain of renal proximal tubules, where they may function together in the urinary excretion of organic anions. Previous reports showed that urinary excretion of some organic anions is not impaired in transport-deficient (TR⁻) rats, which lack Mrp2, suggesting that up-regulation of other transporter(s) may compensate for the loss of Mrp2 function. The purpose of this study was to determine whether Mrp4 expression in kidney is altered in TR⁻ rats. Mrp4 mRNA expression was quantified using the high-throughput branched DNA signal amplification assay. Mrp4 protein expression was determined by Western blot and immunohistochemical analysis. Mrp4 mRNA in kidney of TR⁻ rats was 100% higher than normal Wistar rats. Western blot analysis showed a 200% increase in Mrp4 protein expression in kidney of the mutant rats compared to normal rats. Immunohistochemical analysis of Mrp4 protein demonstrated apical localization of Mrp4 on renal proximal tubules, and that the immunoreactivity was more intense in kidney sections from TR⁻ rats than those from normal rats. In summary, the results of the present study demonstrate that renal Mrp4 expression is up-regulated in TR⁻ rats, which may explain why urinary excretion of some organic anions remains normal in the mutant rats.

Keywords: Abbreviations; Mrp; multidrug resistance-associated protein; TR; ⁻; transport-deficient rat; UGT; UDP-glucuronosyltransferase; bDNA; branched DNA; PAH; p; -aminohippurate; CYP; cytochrome P450Mrp4; Mrp2; TR; ⁻; rat; Kidney; Up-regulation; Gunn rat

CYP2C-catalyzed delta(9)-tetrahydrocannabinol metabolism: Kinetics, pharmacogenetics and interaction with phenytoin by Tina M. Bland; Robert L. Haining; Timothy S. Tracy; Patrick S. Callery (pp. 1096-1103).

delta(9) -Tetrahydrocannabinol (Δ⁹-THC), the primary psychoactive constituent of marijuana, is subject to first pass hepatic metabolism primarily by hydroxylation to yield active and inactive oxygenated products. The primary metabolite is formed via oxidation of the allylic methyl group to yield 11-hydroxy-Δ⁹-THC, which is oxidized further to 11-nor-9-carboxy-Δ⁹-THC. The hydroxylation is thought to be mediated by CYP2C9. The present study was designed to address the kinetics and pharmacogenetics of CYP2C-mediated metabolism of (Δ⁹)-THC by studying its metabolism in human liver microsomes and expressed enzymes. Expressed CYP2C9.1 exhibited high affinity for the hydroxylation of Δ⁹-THC (apparent K_m, 2μM), similar to that observed in human liver microsomes (apparent K_m, 0.8μM). In contrast, the calculated intrinsic clearance (apparent V_m/ K_m) for CYP2C9.2 and CYP2C9.3 was approximately 30% that of the wild type, CYP2C9.1. Given the high affinity of CYP2C9 for the hydroxylation of Δ⁹-THC, we evaluated the potential for an interaction between Δ⁹-THC, 11-hydroxy-Δ⁹-THC, or 11-nor-9-carboxy-Δ⁹-THC and the CYP2C9 substrate, phenytoin. Surprisingly, Δ⁹-THC increased the rate of phenytoin hydroxylation in human liver microsomes and expressed CYP2C9 enzyme. Similar increases in rate were observed with co-incubation of 11-hydroxy-Δ⁹-THC and 11-nor-9-carboxy-Δ⁹-THC with phenytoin. These in vitro data suggest the potential for an interaction from the concomitant administration of Δ⁹-THC and phenytoin that could result in decreased phenytoin concentrations in vivo.

Keywords: CYP2C; Tetrahydrocannabinol; Phenytoin; Drug interaction; Drug metabolism; Pharmacogenetics

Human organic anion transporter hOAT3 is a potent transporter of cephalosporin antibiotics, in comparison with hOAT1 by Harumasa Ueo; Hideyuki Motohashi; Toshiya Katsura; Ken-ichi Inui (pp. 1104-1113).

We examined the substrate specificity of human organic anion transporter (hOAT) 1 and hOAT3 for various cephalosporin antibiotics, cephaloridine, cefdinir, cefotiam, ceftibuten, cefaclor, ceftizoxime, cefoselis and cefazolin by using HEK293 cells stably transfected with hOAT1 or hOAT3 cDNA (HEK-hOAT1, HEK-hOAT3). Additionally, we examined the uptake of various compounds by these transfectants. The mRNA level of hOAT3 in HEK-hOAT3 was about three-fold that of hOAT1 in HEK-hOAT1. Functional expression of hOAT1 and hOAT3 was confirmed by the uptake of p-[¹⁴C]aminohippurate and [³H]estrone sulfate, respectively. p-[¹⁴C]Aminohippurate, [³H]estrone sulfate, [¹⁴C]captopril, [³H]methotrexate, [³H]ochratoxin A, [³H]leucovorin and [³H]cimetidine were shown to be substrates for hOAT1 and hOAT3, and [³H]dehydroepiandrosterone sulfate was shown to be a substrate for hOAT3. All cephalosporin anitibiotics tested were shown to inhibit the uptake of p-[¹⁴C]aminohippurate and [³H]estrone sulfate via hOAT1 and hOAT3, respectively, in a dose-dependent manner, and the IC₅₀ values of these antibiotics, except for cefaclor, for the hOAT1-mediated uptake of p-[¹⁴C]aminohippurate were within four-fold of those for the hOAT3-mediated uptake of [³H]estrone sulfate. The uptake of cephaloridine, cefdinir and cefotiam by HEK-hOAT3 was 35–50-fold greater than that by control cells. Moreover, the accumulation of the other cephalolsporin antibiotics was significantly greater in HEK-hOAT3 than in control cells. In contrast, the uptake of these antibiotics by HEK-hOAT1 was within two-fold of that by control cells. In conclusion, hOAT3 plays a more important role than hOAT1 in the renal secretion of cephalosporin antibiotics.

Keywords: Abbreviations; hOAT; human organic anion transporter; rOAT; rat organic anion transporterOrganic anion transporter; Cephalosporin antibiotics; p; -Aminohippurate; Estrone sulfate; Renal secretion; Transport

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Biochemical Pharmacology (v.70, #7)