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Biochemical Pharmacology (v.80, #1)

Editorial Advisory Board (pp. iii).

Graphical Abstracts Contents (pp. e1-e5).

Functionally selective cannabinoid receptor signalling: Therapeutic implications and opportunities by Barbara Bosier; Giulio G. Muccioli; Emmanuel Hermans; Didier M. Lambert (pp. 1-12).

The CB₁ and CB₂ cannabinoid receptors are G protein-coupled receptors (GPCRs) recognized by a variety of endogenous ligands and activating multiple signalling pathways. This multiplicity of ligands and intracellular transduction mechanisms supports a complex control of physiological functions by the endocannabinoid system, but requires a finely tuned regulation of the signalling events triggered on receptor activation. Here we review the diverse signalling pathways activated by the cannabinoid receptors and discuss the mechanisms allowing for specificity in the associated functional responses triggered by endogenous or exogenous ligands. At variance with the classical concept that all agonists at a given GPCR induce a similar repertoire of downstream events in all tissues, we also summarize the experimental evidence supporting the existence of functional selectivity and protean agonism at cannabinoid receptors. By placing emphasis on the ligand- or constitutive activity-dependent specifications of receptor–G protein coupling, these concepts explain how distinct cannabinoid ligands may activate specific downstream mediators. Finally, although both the diversity and specificity in cannabinoid signalling are now established in vitro, few data are available from in vivo studies. Therefore, we conclude this review by examining the experimental evidence supporting the physiological relevance of this complexity in the cannabinoid system. The ability to selectively manipulate physiological functions, through activation of defined signalling cascades, will in all likelihood help in the development of efficacious and safe cannabinoid-based therapeutics for a variety of indications.

Keywords: Cannabinoid receptors; Functional selectivity; G protein-coupled receptors; Cell signalling

New molecular targets for the treatment of osteoarthritis by Maria José Alcaraz; Javier Megías; Isabel García-Arnandis; Victoria Clérigues; Maria Isabel Guillén (pp. 13-21).

Osteoarthritis (OA) is a chronic degenerative joint disorder characterized by destruction of the articular cartilage, subchondral bone alterations and synovitis. Current treatments are focused on symptomatic relief but they lack efficacy to control the progression of this disease which is a leading cause of disability. Therefore, the development of effective disease-modifying drugs is urgently needed. Different initiatives are in progress to define the molecular mechanisms involved in the initiation and progression of OA. These studies support the therapeutic potential of pathways relevant in joint metabolism such as Wnt/β-catenin, discoidin domain receptor 2 or proteinase-activated receptor 2. The dysregulation in cartilage catabolism and subchondral bone remodeling could be improved by selective inhibitors of matrix metalloproteinases, aggrecanases and other proteases. Another approach would favor the activity of anabolic processes by using growth factors or regulatory molecules. Recent studies have also revealed the role of oxidative stress and synovitis in the progression of this disease, supporting the development of a number of inhibitory strategies. Novel targets in OA are represented by genes involved in OA pathophysiology discovered using gene network, epigenetic and microRNA approaches. Further insights into the molecular mechanisms involved in OA initiation and progression may lead to the development of new therapies able to control joint destruction and repair.

Keywords: Abbreviations; ADAMTS; a disintegrin and metalloproteinase with thrombospondin motifs; APC; adenomatous polyposis coli; bFGF; basic fibroblast growth factor; BMP; bone morphogenetic protein; CaPPS; calcium pentosan polysulfate; CK1α; casein kinase 1α; CORM-2; tricarbonyldichlororuthenium(II) dimer; DDR2; discoidin domain receptor 2; Dkk; Dikkopf related proteins; ECM; extracellular matrix; ERK; extracellular signal-regulated kinase; FRZB; frizzled related protein; GSK-3β; glycogen synthase kinase 3β; HDAC; histone deacetylase; Hh; hedgehog; HIF-1α; hypoxia-inducible factor 1 α; HMGB2; high mobility group box 2; HO-1; heme oxygenase-1; IGF-1; insulin-like growth factor-1; IGFBP; IGF binding protein; IκBα; inhibitor of κB; IL; interleukin; IL-1Ra; IL-1 receptor antagonist; JNK; c-Jun N-terminal kinase; LAP; latency-associated peptide; LRP; low-density lipoprotein receptor; LTBP; latent TGFβ binding proteins; MAPK; mitogen-activated protein kinase; MMP; matrix metalloproteinase; NF-κB; nuclear factor κB; OA; osteoarthritis; PAR-2; proteinase-activated receptor-2; PDGF; platelet-derived growth factor; PI3-K; phosphoinositide 3-kinase; RANKL; receptor activator for NF-κB ligand; ROS; reactive oxygen species; Runx2; runt-related transcription factor 2; SFRP; secreted frizzled related protein; SLRP; small leucine-rich repeat proteoglycan; Smurf; Smad–ubiquitin regulatory factor; SOD; superoxide dismutase; SOST; sclerostatin; TCF/LEF; resident lymphoid enhancer factor/T-cell; TIMP; tissue inhibitor of metalloproteinases; TNFα; tumor necrosis factor-α; VEGF; vascular endothelial growth factor; WISP-1; Wnt-induced secreted protein-1; TGFβ; transforming growth factor-βOsteoarthritis; Growth factors; Wnt/β-catenin; Cytokines; MicroRNA

Dihydroartemisinin accelerates c-MYC oncoprotein degradation and induces apoptosis in c-MYC-overexpressing tumor cells by Jin-Jian Lu; Ling-Hua Meng; Uma T. Shankavaram; Cai-Hua Zhu; Lin-Jiang Tong; Guang Chen; Li-Ping Lin; John N. Weinstein; Jian Ding (pp. 22-30).

Artemisinin and its derivatives (ARTs) are effective antimalarial drugs and also possess profound anticancer activity. However, the mechanism accounted for its distinctive activity in tumor cells remains unelucidated. We computed Pair wise Pearson correlation coefficients to identify genes that show significant correlation with ARTs activity in NCI-55 cell lines using data obtained from studies with HG-U133A Affymetrix chip. We found c-myc is one of the genes that showed the highest positive correlation coefficients among the probe sets analyzed ( r=0.585, P<0.001). Dihydroartemisinin (DHA), the main active metabolite of ARTs, induced significant apoptosis in HL-60 and HCT116 cells that express high levels of c-MYC. Stable knockdown of c-myc abrogated DHA-induced apoptosis in HCT116 cells. Conversely, forced expression of c-myc in NIH3T3 cells sensitized these cells to DHA-induced apoptosis. Interestingly, DHA irreversibly down-regulated the protein level of c-MYC in DHA-sensitive HCT116 cells, which is consistent to persistent G1 phase arrest induced by DHA. Further studies demonstrated that DHA accelerated the degradation of c-MYC protein and this process was blocked by pretreatment with the proteasome inhibitor MG-132 or GSK 3β inhibitor LiCl in HCT116 cells. Taken together, ARTs might be useful in the treatment of c-MYC-overexpressing tumors. We also suggest that c-MYC may potentially be a biomarker candidate for prediction of the antitumor efficacies of ARTs.

Keywords: Abbreviations; Act D; actinomycin D; ART; artesunate; ARTs; artemisinin and its derivatives; CHX; cycloheximide; DFO; deferoxamine mesylate salt; DHA; dihydroartemisinin; GSK 3β; glycogen-synthase kinase 3β; MB; MYC box; MTT; tetrazolium bromide; NOC; nocodazole; 4-OHT; 4-hydroxy-tamoxifen; PI; propidium iodide; shRNA; short hairpin RNA; SRB; sulforhodamin B; TERT; telomerase reverse transcriptaseDihydroartemisinin; NCI-60; c-MYC; Apoptosis; GSK 3β

Sequential treatment with flavopiridol synergistically enhances pyrrolo-1,5-benzoxazepine-induced apoptosis in human chronic myeloid leukaemia cells including those resistant to imatinib treatment by Sandra A. Bright; Giuseppe Campiani; Michael W. Deininger; Mark Lawler; D. Clive Williams; Daniela M. Zisterer (pp. 31-38).

The Bcr-Abl kinase inhibitor, imatinib mesylate, is the front line treatment for chronic myeloid leukaemia (CML), but the emergence of imatinib resistance has led to the search for alternative drug treatments and the examination of combination therapies to overcome imatinib resistance. The pro-apoptotic PBOX compounds are a recently developed novel series of microtubule targeting agents (MTAs) that depolymerise tubulin. Recent data demonstrating enhanced MTA-induced tumour cell apoptosis upon combination with the cyclin dependent kinase (CDK)-1 inhibitor flavopiridol prompted us to examine whether this compound could similarly enhance the effect of the PBOX compounds. We thus characterised the apoptotic and cell cycle events associated with combination therapy of the PBOX compounds and flavopiridol and results showed a sequence dependent, synergistic enhancement of apoptosis in CML cells including those expressing the imatinib-resistant T315I mutant. Flavopiridol reduced the number of polyploid cells formed in response to PBOX treatment but only to a small extent, suggesting that inhibition of endoreplication was unlikely to play a major role in the mechanism by which flavopiridol synergistically enhanced PBOX-induced apoptosis. The addition of flavopiridol following PBOX-6 treatment did however result in an accelerated exit from the G2/M transition accompanied by an enhanced downregulation and deactivation of the CDK1/cyclin B1 complex and an enhanced degradation of the inhibitor of apoptosis protein (IAP) survivin. In conclusion, results from this study highlight the potential of these novel series of PBOX compounds, alone or in sequential combination with flavopiridol, as an effective therapy against CML.

Keywords: Abbreviations; Abl; abelson; Bcl-2; B-cell leukaemia/lymphoma 2; Bcr; breakpoint cluster region; CDK; cyclin dependent kinase; CI; combination index; CML; chronic myeloid leukaemia; DTT; dithiothreitol; FACS; fluorescent activated cell sorting analysis; FBS; foetal bovine serum; IAP; inhibitor of apoptosis protein; MTA; microtubule targeting agents; NT; no treatment; PBOX; pyrrolo-1,5-benzoxazepine; PBS; phosphate buffered salineCML; PBOX; Survivin; Flavopiridol; Apoptosis

Inhibition of epidermal growth factor receptor-overexpressing cancer cells by camptothecin, 20-(N,N-diethyl) glycinate by V. Badireenath Konkimalla; Thomas Efferth (pp. 39-49).

Camptothecin, 20-(N,N-diethyl) glycinate exerts preferential cytotoxicity towards EGFR-transfectant cells. G2/M DNA damage checkpoint regulation, aryl hydrocarbon receptor signaling, xenobiotic metabolism and endoplasmatic reticulum stress are involved.The epidermal growth factor receptor (EGFR) represents a prognostic marker for short survival of patients and therapy resistance of tumors. Despite clinical usefulness of EGFR tyrosine kinase inhibitors, resistance can develop. Therefore, there is an urgent need for novel EGFR inhibitors. Camptothecins have been characterized as inhibitors of DNA topoisomerase I (TOP1), although a correlation between TOP1 expression and activity is not well established in clinical biopsies. Hence, other targets may also be relevant. By molecular docking, we found that camptothecin 20-N,N-glycinate (CPTg) and camptothecin (CPT) bind to the same pharmacophore at EGFR as erlotinib, albeit to partly different amino acids. To validate the in silico results, CPT and CPTg were evaluated for their cytotoxic activity and downstream signaling mechanisms in U87MG glioblastoma cell lines transduced with different expression vectors for EGFR. All transduced cell lines were more susceptible to CPTg or CPT than the non-transduced cells, indicating a preferential activity towards EGFR-expressing tumor cells. Microarray-based mRNA hybridizations were performed in treated a non-treated cell lines. Subsets of genes were commonly regulated between the cell lines. By pathway analyses, we ranked canonical pathways according to differential gene expression after drug treatment. The pathways for G2/M DNA damage checkpoint regulation, aryl hydrocarbon receptor signaling, and xenobiotic metabolism and endoplasmatic reticulum stress were top ranked. In conclusion, binding of CPTg and CPT to the erlotinib pharmacophore and preferential cytotoxicity towards EGFR-overexpressing cells indicate susceptibility towards erlotinib-resistant tumors. Multiple mechanisms may account for cytotoxicity of these camptothecins.

Keywords: Chemotherapy; Microarray; Oncogenes; Pharmacology; Pharmacogenomics; Signal transduction; Systems biology

HMBA depolymerizes microtubules, activates mitotic checkpoints and induces mitotic block in MCF-7 cells by binding at the colchicine site in tubulin by Biswa Prasun Chatterji; Mithu Banerjee; Parminder Singh; Dulal Panda (pp. 50-61).

10-[(3-Hydroxy-4-methoxybenzylidene)]-9(10H)-anthracenone (HMBA), a synthetic compound, has been reported to have a potent antitumor activity. In this study, we found that HMBA depolymerized microtubules in MCF-7 cells and produced aberrant spindles in the MCF-7 cells. It also reduced the distance between the centrosomes and activated the mitotic checkpoint proteins BubR1 and Mad2. Further, HMBA inhibited the progression of MCF-7 cells in mitosis and induced apoptotic cell death involving p53 pathway. In vitro, HMBA bound to purified brain tubulin with a dissociation constant of 4.1±0.9μM. It inhibited microtubule assembly and increased the GTP hydrolysis rate of microtubule assembly. The compound did not alter the binding of 2′(or 3′)- O-(trinitrophenyl) guanosine 5′-triphosphate (TNP–GTP), a fluorescent analogue of GTP, to tubulin suggesting that it did not inhibit the binding of GTP to tubulin. However, we obtained evidence indicating that HMBA perturbed the conformation of the GTP binding site in tubulin. In addition, an analysis of the modified Dixon plot suggested that HMBA competitively inhibited the binding of colchicine to tubulin. A computational analysis of the binding of HMBA to tubulin supported the finding that HMBA shared its binding site with colchicine in tubulin and indicated that the binding of HMBA to tubulin was primarily stabilized through hydrogen bonding.

Keywords: Abbreviations; IgG; immunoglobulin G; GTP; guanosine triphosphate; GDP; guanosine diphosphate; FITC; fluorescein isothiocyanate; ANS; 1-anilinonaphthalene-8-sulfonic acid; EGTA; ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; DAMA–colchicine; N-deacetyl-N-(2-mercaptoacetyl)-colchicine; PI; propidium Iodide; PBS; phosphate buffered saline; DMSO; dimethyl sulfoxideMicrotubule; HMBA; Apoptosis; Anticancer drugs; Spindle assembly checkpoint proteins; p53

Induction of p53-independent apoptosis by a novel synthetic hexahydrocannabinol analog is mediated via Sp1-dependent NSAID-activated gene-1 in colon cancer cells by Dinesh Thapa; Dinesh Babu; Min-A. Park; Mi-Kyoung Kwak; Yong-Rok Lee; Jeong Min Kim; Taeg Kyu Kwon; Jung-Ae Kim (pp. 62-71).

Proposed mechanism of action of a hexahydrocannabinol analog LYR-8 which induces p53-independent apoptosis via Sp-1-dependent NAG-1 activation in colon cancer cells.Nonsteroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) has received greater attention as a novel molecular target for anti-cancer therapeutics in recent years. We identified a novel synthetic hexahydrocannabinol analog, LYR-8 [(1-((9 S)-1-hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6 H-benzo[c]chromen-2-yl)ethanone)], as a potent NAG-1 and apoptosis inducer in a panel of human cancer cells. LYR-8 did not possess any affinity for cannabinoid receptor CB₁ or CB₂, which eliminates the concern about potential psychoactive side effects. LYR-8 dramatically induced NAG-1 expression and apoptosis in HCT116 (wild-type p53) and HT29 (mutant p53) colon cancer cells. The NAG-1 expression by LYR-8 was not blocked by pifithrin-α, a specific p53 inhibitor, which was different from doxorubicin that induced p53-dependent NAG-1 transcriptional activity. The induction of NAG-1 promoter activity by LYR-8 was strongly correlated with increased Sp1 activation as noted in various luc-promoter activities. Furthermore, pretreatment with the specific Sp1 inhibitor mithramycin A completely reversed the LYR-8-induced NAG-1 expression in both HCT116 and HT29 cells. Knockdown of NAG-1 using siRNA significantly reversed LYR-8-induced cell death in both wild-type and mutant p53-expressing colon cancer cells. Furthermore, sensitization with NAG-1 inducer sulindac sulfide synergized LYR-8-induced cell death in both colon cancer cells. These results suggest that induction of NAG-1 via Sp1 activation is a promising therapeutic approach in cancer treatment, and that a novel compound like LYR-8 could be a potent chemotherapeutic agent for colon cancers including p53-mutated cancer.

Keywords: Hexahydrocannabinol; NAG-1; Sp1; p53; Colon cancer

Direct assessment of P-glycoprotein efflux to determine tumor response to chemotherapy by Gauri Patwardhan; Vineet Gupta; Juowen Huang; Xin Gu; Yong-Yu Liu (pp. 72-79).

Multidrug resistance is a major impediment to the success of cancer chemotherapy. The overproduced P-glycoprotein that extrudes anticancer drugs from cells, is the most common mechanism detected in multidrug-resistant cancers. Direct measurement of cellular efflux of tumors in vivo, rather than estimation of MDR1 mRNA and P-glycoprotein levels in samples stored or embedded, can functionally characterize the mechanism of drug resistance and determine the choice of anticancer drugs for cancer patients. Herewith, we introduce a new approach to directly determine P-glycoprotein efflux of tumors. Employing Flutax-2 (Oregon green-488 paclitaxel) and fluorescence spectrophotometry, this method has successfully measured cellular transportability including efflux and accumulation in diverse cancer cell lines, tumors and other tissues with high reproducibility. With this method, we have quantitatively determined cellular efflux that is correlated with P-glycoprotein levels and the reversal effects of agents in cell lines of breast, ovarian, cervical and colon cancers, and in tumor-bearing mice. It has sensitively detected these alterations of P-glycoprotein efflux in approximately 5mg tumor or other tissues with high confidence. This direct and quick functional assessment has a potential to determine drug resistance in different types of cancers after surgical resection. Further validation of this method in clinic settings for the diagnosis of drug resistance purpose is needed.

Keywords: Drug resistance; Paclitaxel; Efflux; Accumulation; P-glycoprotein; Fluorescence; Cancer

Troglitazone is an estrogen-related receptor α and γ inverse agonist by Yanfei Wang; Fang Fang; Chi-Wai Wong (pp. 80-85).

Troglitazone functions as an ERRα/γ inverse agonist, suppressing their interactions with PGC-1 coactivators, reducing SOD1/2 expression, and enhancing ROS production to induce p21^WAF/GADD45α expression arresting cell cycle.As a ligand for peroxisome proliferators-activated receptor γ (PPARγ), troglitazone inhibits cell growth by mechanisms besides activating PPARγ. In this study, we found that troglitazone interfered with the interactions between estrogen-related receptor α and γ (ERRα and ERRγ) and their coactivator PPARγ coactivator-1α (PGC-1α) functioning as an inverse agonist. Additionally, troglitazone suppressed the expressions of PGC-1α and its related member PGC-1β which are key regulators of mitochondrial function. Consequently, troglitazone reduced mitochondrial mass and suppressed the expressions of superoxide dismutases to elevate reactive oxygen species (ROS) production. The increase in ROS in turn induced the expression of cell cycle inhibitor p21^WAF1. We therefore propose that ERRα and ERRγ are alternative targets of troglitazone important for mediating its growth suppressive effect.

Keywords: Troglitazone; ERRα; ERRγ; Mitochondrial biogenesis; ROS

An Angiotensin II type 1 receptor activation switch patch revealed through Evolutionary Trace analysis by Marie Mi Bonde; Rong Yao; Jian-Nong Ma; Srinivasan Madabushi; Stig Haunsø; Ethan S. Burstein; Jennifer L. Whistler; Søren P. Sheikh; Olivier Lichtarge; Jakob Lerche Hansen (pp. 86-94).

Seven transmembrane (7TM) or G protein-coupled receptors constitute a large superfamily of cell surface receptors sharing a structural motif of seven transmembrane spanning alpha helices. Their activation mechanism most likely involves concerted movements of the transmembrane helices, but remains to be completely resolved. Evolutionary Trace (ET) analysis is a computational method, which identifies clusters of functionally important residues by integrating information on evolutionary important residue variations with receptor structure. Combined with known mutational data, ET predicted a patch of residues in the cytoplasmic parts of TM2, TM3, and TM6 to form an activation switch that is common to all family A 7TM receptors. We tested this hypothesis in the rat Angiotensin II (Ang II) type 1a (AT1a) receptor. The receptor has important roles in the cardiovascular system, but has also frequently been applied as a model for 7TM receptor activation and signaling. Six mutations: F66A, L67R, L70R, L119R, D125A, and I245F were targeted to the putative switch and assayed for changes in activation state by their ligand binding, signaling, and trafficking properties. All but one receptor mutant (that was not expressed well) displayed phenotypes associated with changed activation state, such as increased agonist affinity or basal activity, promiscuous activation, or constitutive internalization highlighting the importance of testing different signaling pathways. We conclude that this evolutionary important patch mediates interactions important for maintaining the inactive state. More broadly, these observations in the AT1 receptor are consistent with computational predictions of a generic role for this patch in 7TM receptor activation.

Keywords: 7TM receptor; GPCR; Evolutionary Trace; Constitutive activity; Angiotensin

Sphingosylphosphorylcholine down-regulates filaggrin gene transcription through NOX5-based NADPH oxidase and cyclooxygenase-2 in human keratinocytes by Hyun Choi; Shinhyoung Kim; Hyoung-June Kim; Kwang-Mi Kim; Chang-Hoon Lee; Jennifer H. Shin; Minsoo Noh (pp. 95-103).

Sphingosylphosphorylcholine (SPC) mediates various inflammatory and behavioral responses in atopic dermatitis. Recent studies have shown that dysfunction of the epidermal permeability barrier itself plays a primary role in the etiology of atopic dermatitis. However, the effects of SPC on major proteins essential to the development of the epidermal permeability barrier such as filaggrin, loricrin, involucrin, keratin 1, keratin 10 and small proline-rich proteins are still unclear. In this study, we demonstrated that SPC significantly reduces filaggrin gene transcription, implying that SPC plays a pivotal role in impairment of the epidermal permeability barrier in atopic dermatitis lesional skin. In cultured normal human keratinocytes (NHKs), SPC increases the intracellular level of reactive oxygen species (ROS) and up-regulates NADPH oxidase 5 (NOX5) gene transcription. SPC also stimulates prostaglandin (PG) E₂ production by increasing cyclooxygenase (COX)-2 expression in NHK. The effects of the prostanoid EP receptor agonists, limaprost, butaprost, and sulprostone on filaggrin gene expression in NHK suggest that the prostanoid EP2 receptor plays a significant role in the PGE₂-mediated filaggrin down-regulation. In contrast, limaprost and butaprost do not affect NOX5 expression in NHK, implying that the NOX5-regulated ROS pathway stimulated by SPC may be upstream of the COX-2 pathway. We propose that the increase in SPC levels further aggravates dermatological symptoms of atopic dermatitis through SPC-induced down-regulation of filaggrin in NHK.

Keywords: Sphingosylphosphorylcholine; Filaggrin; NADPH oxidase 5; Cyclooxygenase-2; Atopic dermatitis

The signalling role of action potential depolarization in insulin secretion by M. Willenborg; H. Ghaly; K. Hatlapatka; K. Urban; U. Panten; I. Rustenbeck (pp. 104-112).

When K_ATP channels are closed in the absence of glucose TEA still blocks Kv channels and increases cytosolic Ca²⁺, but inhibits insulin secretion. This cannot be overcome by Bay K8644.The K⁺ channel blocker, TEA is known to increase action potential amplitude and insulin secretion of mouse β-cells when added to a nutrient secretagogue. In the presence of a maximally effective sulfonylurea concentration (2.7μM glipizide) the nutrient secretagogue α-ketoisocaproic acid (KIC, 10mM) strongly increased insulin secretion (about elevenfold). Instead of enhancing the effect of KIC, TEA reduced the KIC-induced secretion by more than 50%. Also, the secretion rate produced by 2.7μM glipizide alone was significantly reduced by TEA. In contrast, TEA enhanced the insulinotropic effect of glipizide when a basal glucose concentration (5mM) was present. In the presence as well as in the absence of glucose glipizide produced a plateau depolarization with superimposed action potentials. Under both conditions, TEA increased the glipizide-induced action potential amplitude and further elevated the cytosolic free calcium concentration ([Ca²⁺]_i) with an oscillatory characteristic. These effects depended on the activity of L-type Ca²⁺ channels, even though the effect of TEA differed from that of 1μM of the Ca²⁺ channel opener, Bay K8644, which primarily increased action potential duration. TEA did not negatively affect parameters of β-cell energy metabolism (NAD(P)H fluorescence and ATP/ADP ratio), rather, it slightly increased NAD(P)H fluorescence. Apparently, TEA inhibits insulin secretion in the absence of glucose in spite of a persistent ability to block K⁺ ion conductance. Thus, the signalling role of action potential depolarization in insulin secretion may require reconsideration and ion conductance-independent actions of K⁺ channels may be involved in this paradox effect of TEA.

Keywords: Abbreviations; [Ca; ²⁺; ]; _i; cytosolic free calcium concentration; Kv channel; voltage-dependent K; ⁺; channel; K; _ATP; channel; ATP-sensitive K; ⁺; channel; KIC; α-ketoisocaproic acid; TEA; tetraethylammoniumCytosolic calcium concentration; Insulin secretion; Kv channel; K; _ATP; channel; Pancreatic islets; Plasma membrane potential

NPPB structure-specifically activates TRPA1 channels by Kun Liu; Manoj Samuel; Melisa Ho; Richard K. Harrison; Jeff W. Paslay (pp. 113-121).

TRPA1 channels have been found to play an important role in mammalian pain sensation, especially when the pain is caused by chemicals on site of inflammation. A large number of structurally diverse chemicals are found to activate TRPA1 channels, implicating a potential chemosensor in neuronal nociception. Identification of the channel activation by cysteine modification through covalent chemical reaction provides arguments for the diversity of the agonist structures. However, it is largely unknown how nonreactive compounds activate TRPA1 channels. Here, we report that NPPB, a classic Cl⁻ channel blocker, potently activated human TRPA1 channels overexpressed in mammalian HEK-293 cells. This effect was confirmed in Ca²⁺ imaging assay, patch clamp whole cell and single channel recordings. The NPPB response was quick, fully reversible and replicable, contrary to the effect of covalent modification by AITC. The mutagenesis studies revealed a refreshed look at several mutations known to be critical for the actions of AITC and menthol. The blocking profile of NPPB on these mutants showed that the NPPB activation was similar to that of FTS and different from AITC and menthol. The results indicated a possible close interaction between S5 and N-terminal domains of the channel. We also tested a group of NPPB analogs on TRPA1 channel activities. The results demonstrated that NPPB activation was tightly associated with chemical structure. None of the single chemical group was sufficient to activate the channel, indicating that NPPB activated TRPA1 through a structure-specific mechanism.

Keywords: Abbreviations; NPPB; 5-nitro-2-(3-phenylpropylamino)benzoic acid; TRP; transient receptor potential; ROS; reactive oxygen species; AITC; allyl isothiocyanate; FTS; farnesyl thiosalicylic acid; CFTR; cystic fibrosis transmembrane conductance regulator; FLIPR; Fluometric Imaging Plate ReaderTRPA1 channels; NPPB; Covalent modification; Structure-specific modification; Mutagenesis

Effects of monoamine oxidase inhibitor and cytochrome P450 2D6 status on 5-methoxy- N, N-dimethyltryptamine metabolism and pharmacokinetics by Hong-Wu Shen; Chao Wu; Xi-Ling Jiang; Ai-Ming Yu (pp. 122-128).

5-Methoxy- N, N-dimethyltryptamine (5-MeO-DMT) is a natural psychoactive indolealkylamine drug that has been used for recreational purpose. Our previous study revealed that polymorphic cytochrome P450 2D6 (CYP2D6) catalyzed 5-MeO-DMT O-demethylation to produce active metabolite bufotenine, while 5-MeO-DMT is mainly inactivated through deamination pathway mediated by monoamine oxidase (MAO). This study, therefore, aimed to investigate the impact of CYP2D6 genotype/phenotype status and MAO inhibitor (MAOI) on 5-MeO-DMT metabolism and pharmacokinetics. Enzyme kinetic studies using recombinant CYP2D6 allelic isozymes showed that CYP2D6.2 and CYP2D6.10 exhibited 2.6- and 40-fold lower catalytic efficiency ( V_max/ K_m), respectively, in producing bufotenine from 5-MeO-DMT, compared with wild-type CYP2D6.1. When co-incubated with MAOI pargyline, 5-MeO-DMT O-demethylation in 10 human liver microsomes showed significantly strong correlation with bufuralol 1′-hydroxylase activities ( R²=0.98; P<0.0001) and CYP2D6 contents ( R²=0.77; P=0.0007), whereas no appreciable correlations with enzymatic activities of other P450 enzymes. Furthermore, concurrent MAOI harmaline sharply reduced 5-MeO-DMT depletion and increased bufotenine formation in human CYP2D6 extensive metabolizer hepatocytes. In vivo studies in wild-type and CYP2D6-humanized (Tg- CYP2D6) mouse models showed that Tg- CYP2D6 mice receiving the same dose of 5-MeO-DMT (20mg/kg, i.p.) had 60% higher systemic exposure to metabolite bufotenine. In addition, pretreatment of harmaline (5mg/kg, i.p.) led to 3.6- and 4.4-fold higher systemic exposure to 5-MeO-DMT (2mg/kg, i.p.), and 9.9- and 6.1-fold higher systemic exposure to bufotenine in Tg- CYP2D6 and wild-type mice, respectively. These findings indicate that MAOI largely affects 5-MeO-DMT metabolism and pharmacokinetics, as well as bufotenine formation that is mediated by CYP2D6.

Keywords: Abbreviations; CYP2D6; cytochrome P450 2D6; 5-MeO-DMT; 5-methoxy-; N; ,; N; -dimethyltryptamine; MAOI; monoamine oxidase inhibitor; DDI; drug–drug interactions; EM; extensive metabolizer; PM; poor metabolizer; HLM; human liver microsomes; HPLC; high performance liquid chromatography; LC–MS/MS; liquid chromatography tandem mass spectrometryCYP2D6; 5-MeO-DMT; MAOI; Pharmacokinetics; Transgenic mouse

Constitutive androstane/active receptor is a target of retinoic acid receptor in humans by Kosuke Saito; Kaoru Kobayashi; Yuki Mizuno; Tomomi Furihata; Kan Chiba (pp. 129-135).

Nuclear receptor constitutive androstane/active receptor (CAR) is well known as a transcription factor regulating many genes that encode drug-metabolizing enzymes and factors modulating hepatic gluconeogenesis. However, there have been few studies on regulation of the CAR gene itself. In this study, we examined the involvement of retinoic acid receptor α (RARα) in transcriptional regulation of the CAR gene in the liver. The expression levels of CAR mRNA in human primary hepatocytes and HepG2 cells were increased by all-trans retinoic acid. Activities of the human CAR promoter containing a region (termed cRARE) located at +1453/+1469 within intron 1 were increased by co-expression of RARα in HepG2 cells. In addition, introduction of mutation into cRARE abolished transcriptional activation of the promoter by RARα. The results of gel mobility shift assay and chromatin immunoprecipitation assay showed that RARα was bound to cRARE. These results suggest that RARα transactivated the human CAR gene by binding to cRARE located at +1453/+1469 within intron 1 of the gene. In contrast, the rat CAR gene was not activated by exposure to all-trans retinoic acid, probably due to the lack of a region corresponding to cRARE in the human CAR gene. Although the physiological significance of RARα-dependent up-regulation of CAR in the human liver remains to be clarified, retinoid metabolism may be regulated by the up-regulation of CAR.

Keywords: Abbreviations; atRA; all-trans retinoic acid; CAR; constitutive androstane/active receptor; ChIP; chromatin immunoprecipitation; cRARE; CAR RARα response element; CYP; cytochrome P450; FoxO1; forkhead transcription factor 1; IRS; insulin response sequence; PEPCK; phosphoenolpyruvate carboxykinase; RAR; retinoic acid receptor; RXR; retinoid X receptorConstitutive androstane/active receptor (CAR); Retinoic acid receptor (RAR); All-trans retinoic acid; Nuclear receptor; Intron; Gene regulation

Biochemical characterization of the binding of cyclic RGDyK to hepatic stellate cells by Xiao-wei Huang; Ji-Yao Wang; Feng Li; Zheng-Ji Song; Cao Xie; Wei-Yue Lu (pp. 136-143).

Activated hepatic stellate cells (HSCs) play a crucial role in the development of liver fibrosis. Noninvasive monitoring of the activation of HSCs has been challenging due to the lack of specific receptors or motifs on the cells. The present study provides the evidence that integrin αvβ3 expressed on HSCs is a biomarker reflecting the activation of HSCs. Solid-phase synthesis of cRGDyK (Arg-Gly-Asp-_DTyr-Lys) peptide and FAM-conjugated peptide were employed for binding to integrin αvβ3. The increased expression of integrin αv and β3 at mRNA and protein levels was detected during HSC activation. The affinity of cRGDyK to integrin αvβ3 was examined by both radioligand binding assay and FAM-conjugated peptide binding measurements. Quantitative RT-PCR and Western blotting showed a less dramatic, but significant increase in αv and β3 integrin mRNA and protein expression following activation of rat HSCs. Radioiodinated cRGDyK binds to both purified and membrane-bound integrin αvβ3 with high affinity in a dissociable manner. FAM-conjugated cRGDyK was coupled to activated HSCs in a time- and dose-dependent, receptor-mediated manner. Activated HSCs express sufficient number of integrin αvβ3 receptor. cRGDyK peptide binds to both purified and membrane-bound integrin αvβ3 with high affinity in a reversible fashion. Thus, the cRGDyK peptide represented a new agent potentially useful for the diagnosis of liver fibrosis.

Keywords: Abbreviations; HSC; hepatic stellate cell; DMEM; Dulbecco's minimal Eagle's medium; FBS; fetal bovine serum; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; Ct; cycle threshold; PBS; phosphate-buffered saline; ECM; extracellular matrix; cRGDyK; cyclo-(Arg-Gly-Asp-; d; Tyr-Lys); FAM; carboxyfluorescein; cRGAyK; cyclo-(Arg-Gly-Ala-; d; Tyr-Lys); DAPI; 4,6-diamidino-2-phenylindole; RT-PCR; reverse transcriptase-polymerase chain reactionLiver fibrosis; Hepatic stellate cell; Integrin αvβ3; cRGDyK peptide; Targeted binding

Induction of the Nrf2-driven antioxidant response by tert-butylhydroquinone prevents ethanol-induced apoptosis in cranial neural crest cells by Dong Yan; Jian Dong; Kathleen K. Sulik; Shao-yu Chen (pp. 144-149).

Previous studies have shown that ethanol exposure causes apoptosis in cranial neural crest cells (NCCs), an ethanol-sensitive cell population implicated in Fetal Alcohol Spectrum Disorders (FASD). Additionally, induction of endogenous antioxidants through activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) has been shown to prevent oxidative stress and apoptosis in ethanol-exposed mouse embryos. The objective of this study was to test whether tert-butylhydroquinone (tBHQ), an Nrf2 inducer, can protect NCCs against ethanol-induced apoptosis. Ethanol exposure was shown to cause a moderate increase in the protein expression of Nrf2 and its downstream antioxidants in the NCCs. Treatment of NCCs with tBHQ alone significantly increased the protein expression of Nrf2 and its downstream antioxidants and also significantly increased the activities of the antioxidant enzymes. In NCCs exposed to ethanol, the tBHQ-mediated antioxidant response prevented oxidative stress and apoptosis. These results clearly demonstrate that the activation of Nrf2 signaling confers protection against ethanol-induced apoptosis in NCCs.

Keywords: Neural crest cells; Nrf2; Ethanol; Antioxidant; Apoptosis

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Biochemical Pharmacology (v.80, #1)

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Biochemical Pharmacology (v.80, #1)