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Biochemical Pharmacology (v.80, #4)

Editorial Advisory Board (pp. iii).

Graphical Abstracts Contents (pp. e1-e4).

Apoptosis and cancer stem cells: Implications for apoptosis targeted therapy by Frank A.E. Kruyt; Jan Jacob Schuringa (pp. 423-430).

Evidence is accumulating showing that cancer stem cells or tumor-initiating cells are key drivers of tumor formation and progression. Successful therapy must therefore eliminate these cells, which is hampered by their high resistance to commonly used treatment modalities. Thus far, only a limited number of studies have addressed the cancer stem cell killing potential of apoptosis targeted therapies and mechanisms of apoptosis resistance in these cells. Apoptosis resistance may involve inherent cellular mechanisms that may change depending on the differentiations status of stem cells and, on the other hand, extrinsic factors provided by the microenvironment such as secreted survival factors, adhesion-mediated apoptosis resistance and hypoxic conditions. In order to metastasize, cancer stem cells from solid tumors have to break free from their primary epithelial sites and resist cell death activation after detachment (anoikis). The induction of an embryonic genetic program causing the transition from an epithelial to a mesenchymal state (EMT) has been implicated in enhanced migration and metastatic spread of tumor cells and may contribute to apoptosis and anoikis resistance. Considering the plasticity of cancer stem cells the question arises whether a particular apoptosis-inducing strategy will be sufficient for eliminating all the cellular appearances of these cells, also taking into account a varying microenvironment. Here, the different mechanisms of apoptosis resistance that may be encountered in the context of cancer stem cell plasticity described thus far are discussed in relation to the efficacy of apoptosis therapies, such as TRAIL, BCL-2 family and XIAP targeted therapies.

Keywords: Tumor-initiating cells; Resistance; Epithelial–mesenchymal transition; Microenvironment; Anoikis; Plasticity

Role of eicosanoids on intestinal epithelial homeostasis by Rut Ferrer; Juan J. Moreno (pp. 431-438).

The intestinal epithelium is a highly dynamic system that is continuously renewed by a process involving cell proliferation and differentiation. Moreover, it is the main interface with the external environment, and maintenance and regulation of the epithelial structure and epithelial barrier function are key determinants of digestive health and host well being. The tight junction, a multiprotein complex composed of transmembrane proteins associated with the cytoskeletal peri-junctional ring of actin and myosin, is an essential component of this barrier that is strictly regulated in a spatio-temporal manner by a complex signaling network. Defects in the intestinal epithelial barrier function have been observed in inflammatory bowel disease, and a classic example of the connection between inflammation and cancer is the increased risk of colorectal cancer in patients with inflammatory bowel disease. In recent years, several molecules have emerged as critical players contributing to inflammation-associated colorectal cancer. For example, eicosanoids derived from arachidonic acid are proposed as mediators involved in the regulation of epithelial structure/function. Interestingly, the tissue concentration of eicosanoids increases during mucosal inflammation and colorectal cancer development. This overview focuses on the physiological and physiopathological roles of eicosanoids in cell growth/cell differentiation/apoptosis and in the paracellular permeability of the intestinal epithelium. A better understanding of these processes will foster new ideas for the development of therapies for these chronic disorders.

Keywords: Abbreviations; AA; arachidonic acid; COX; cyclooxygenase; EETs; epoxyeicosatrienoic acids; EGF; epidermal growth factor; HETEs; hydroxyeicosatetraenoic acids; HODE; hydroxyoctadecadienoic acids; IBD; inflammatory bowel disease; LOX; lipoxygenase; LTs; leukotrienes; PSC; prostaglandin-expressing stromal cells; PGs; prostaglandins; cPLA; ₂; cytosolic phospholipase A; ₂; iPLA; ₂; calcium-independent phospholipase A; ₂; sPLA; ₂; secreted phospholipase A; ₂; PPAR; peroxisome proliferator-activated receptor; NSAIDs; non-steroidal anti-inflammatory drugs; TJ; tight junction; ZO; zonula occludensProstaglandin; Leukotriene; Hydroxyeicosatetraenoic acid; Cell proliferation; Cell differentiation; Barrier function

Tyrosine phosphatase inhibition triggers sustained canonical serine-dependent NFκB activation via Src-dependent blockade of PP2A by Sandra Barisic; Claudia Schmidt; Henning Walczak; Dagmar Kulms (pp. 439-447).

Tyrosine phosphatase inhibition by orthovanadate (OVA) triggers sustained canonical serine-dependent NFκB activation via Src-dependent blockade of the serine/threonine phosphatase PP2A.Activation status of Tyr-kinase Src as well as of the transcription factor NFκB is a decisive criterion for the onset of cancer and in conveying radio-resistance. While the activation status of Src is Tyr phosphorylation-dependent, NFκB activation requires Ser phosphorylation of its cytosolic inhibitor, IκBα. Since constitutive NFκB activation was linked to tumor maintenance, its tight regulation is mandatory.We provide evidence that inhibition of pan-Tyr phosphatase activity by orthovanadate is translated via Src to inhibition of Ser phosphatase PP2A, thereby changing the physiologic response of the cell. In particular we unravelled a new sequence of molecular interactions linking initial activating Tyr416 phosphorylation of Src not to Tyr42-dependent phosphorylation and degradation of IκBα, but to sustained Ser177/181 phosphorylation of IκBα kinase IKKβ following IL-1 stimulation. Consequently, sustained IKKβ activation provides for chronic canonical IκBα degradation, thereby eliciting constitutive NFκB activation. As the critical translator of Tyr to Ser phosphorylation we identified Ser/Thr phosphatase PP2A. We show that the catalytic subunit PP2Ac serves as a Src substrate with Tyr307 phosphorylation leading to its catalytic inhibition. Additionally to the known survival pathways triggered by Src, Src-mediated canonical and persistent NFκB activation may fortify its tumorigenic effects.

Keywords: IKKβ; IκBα; NFκB; PP2A; Src-kinase; Interleukin-1

The role of the N-terminus of mammalian copper transporter 1 in the cellular accumulation of cisplatin by Christopher A. Larson; Preston L. Adams; Danielle D. Jandial; Brian G. Blair; Roohangiz Safaei; Stephen B. Howell (pp. 448-454).

The mammalian copper transporter 1 (CTR1) is responsible for the uptake of copper (Cu) from the extracellular space, and has been shown to play a major role in the initial accumulation of platinum-based drugs. In this study we re-expressed wild type and structural variants of hCTR1 in mouse embryo fibroblasts in which both alleles of mCTR1 had been knocked out (CTR1^−/−) to examine the role of the N-terminal extracellular domain of hCTR1 in the accumulation of cisplatin (cDDP). Deletion of either the first 45 amino acids or just the⁴⁰MXXM⁴⁵ motif in the N-terminal domain did not alter subcellular distribution or the amount of protein in the plasma membrane but it eliminated the ability of hCTR1 to mediate the uptake of Cu. In contrast it only partially reduced cDDP transport capacity. Neither of these structural changes prevented cDDP from triggering the rapid degradation of hCTR1. However, they did alter the potency of the cDDP that achieved cell entry, possibly reflecting the fact that hCTR1 may mediate the transport of cDDP both through the pore it forms in the plasma membrane and via endocytosis. We conclude that cDDP interacts with hCTR1 both at⁴⁰MXXM⁴⁵ and at sites outside the N-terminal domain that produce the conformational changes that trigger degradation.

Keywords: Abbreviations; cDDP; cisplatin; CTR1; copper transporter 1; ICP-MS; inductively coupled plasma mass spectrometry; ICP-OES; inductively coupled plasma optical emission spectroscopy; PBS; phosphate buffered saline; TBS; tris buffered salineCisplatin; Copper transporter 1; Cytotoxicity; Structure; Transport

The IGFR1 inhibitor NVP-AEW541 disrupts a pro-survival and pro-angiogenic IGF-STAT3-HIF1 pathway in human glioblastoma cells by Marzia B. Gariboldi; Raffaella Ravizza; Elena Monti (pp. 455-462).

An autocrine circuit, involving IGF1R, STAT3, HIF1α and IGF-II operates in human glioblastoma cells.Inappropriate activation of the IGF (insulin-like growth factor) system has been implicated in the growth and progression of a number of tumor types. Recent evidence indicates a possible role for the IGF system in modulating/mediating tumor cell response to hypoxia, a common occurrence in solid tumors, and particularly in malignant gliomas, causing tumor cells either to die, or to mount a pleiotropic adaptive response that is mainly orchestrated through activation of the hypoxia-inducible transcription factor HIF1. Experimental evidence suggests possible links between IGF- and HIF1-dependent signaling pathways, as well as a role for activated STAT3 in mediating their activities. Interestingly, igf2 is among the target genes transactivated by HIF1, thereby providing the missing link in a hypothetical autocrine self-amplifying circuit.The present study investigates the presence of the IGF-HIF1-VEGF axis in the human glioma cell line U-87 MG, and characterizes its molecular effectors. Our results show that exogenous IGF-I causes IGF1R and STAT3 activation, and increases HIF1α protein levels and HIF1 trascriptional activity, inducing VEGF release; a similar response, mediated by IGF-II release, is observed following HIF1α stabilization. The existence of an autocrine loop is confirmed by its down-regulation following inactivation of IGF1R (using the IGF1R-specific tyrosine kinase inhibitor NVP-AEW541), STAT3 (transfecting the cells with an expression vector encoding a dominant negative form of STAT3), or HIF1 (using the small molecule inhibitor YC-1). The ability of NVP-AEW541 to block this circuit could be beneficial in suppressing the growth and angiogenic potential of hypoxic glial tumors.

Keywords: Insulin-like growth factors; Hypoxia-inducible factor-1; STAT3; Glioblastoma; IGF-1 receptor inhibitor

Up-regulation of connexin 32 gene by 5-aza-2′-deoxycytidine enhances vinblastine-induced cytotoxicity in human renal carcinoma cells via the activation of JNK signalling by Y. Takano; H. Iwata; Y. Yano; M. Miyazawa; N. Virgona; H. Sato; K. Ueno; T. Yano (pp. 463-470).

Enforced expression of connexin (Cx) 32 gene, a member of gap junction gene family and a tumor suppressor gene in human renal cell carcinoma (RCC), enhanced vinblastine (VBL)-induced cytotoxicity on RCC cells, due to the suppression of multidrug resistance 1 ( MDR1) gene product, P-glycoprotein (P-gp). Also, Cx32 gene in RCC is silenced by hypermethylation of CpG islands in a promoter region of the Cx gene. In this study, we investigated if a DNA demethylating agent, 5-aza-2′-deoxycytidine (5-Aza) could enhance susceptibility of RCC cells (Caki-1) to VBL. We found that 5-Aza treatment up-regulated Cx32 in Caki-1 cells, and the induction of the Cx led to the suppression of P-gp through inhibition of Src and subsequent activation of c-Jun NH₂-terminal kinase (JNK). Moreover, increased transcription activity of c-Jun by the JNK activation contributed to the down-regulation of MDR1, thus indicating a central role of JNK signalling to suppress P-gp level in 5-Aza-treated Caki-1 cells. Chemical sensitivity to VBL in Caki-1 cells was increased by 5-Aza pre-treatment, and this effect was abrogated by short interfering RNA (siRNA)-mediated knockdown of Cx32. Furthermore, co-treatment of 5-Aza or a P-gp inhibitor with VBL drastically enhanced JNK activation comparing to only VBL treatment in Caki-1 cells. These results suggest that the restoration of Cx32 by 5-Aza pre-treatment improves chemical tolerance on VBL in Caki-1 cells and that the JNK activation is a key factor to induce the effect.

Keywords: Connexin32; Renal cell carcinoma; Caki-1 cells; 5-Aza-2′-deoxycytidine; JNK

Chrysin blocks topotecan-induced apoptosis in Caco-2 cells in spite of inhibition of ABC-transporters by Marco Schumacher; Anja Hautzinger; Annette Rossmann; Susanne Holzhauser; Daniela Popovic; Anke Hertrampf; Sabine Kuntz; Michael Boll; Uwe Wenzel (pp. 471-479).

Chrysin potently inhibits topotecan-triggered apoptosis in Caco-2 cells in spite of inhibition of ATP-dependent efflux transporters and comcomitantly increased intracellular drug accumulation by inhibition of caspases and stabilization of β-catenin.ATP-driven efflux pumps such as phosphoglycoprotein-170 (P-gp), multidrug-resistance-associated protein-2 (MRP-2), or breast cancer resistance protein (BCRP) play a crucial role in limiting the efficacy of tumor pharmacotherapy. Selected flavonoids have been suggested to inhibit individual efflux-transporters and to act therefore as multidrug-resistance reversing agents. In the present study it is shown that the flavonoid chrysin acts as a potent inhibitor of P-gp, MRP-2, and BCRP in Caco-2 colon carcinoma cells. As a consequence, cells accumulated higher rates of the apoptosis-inducing chemotherapeutic topotecan in the presence of chrysin, even though under these conditions the expression of the transporters was markedly increased. Interestingly, in spite of the enhanced cellular drug accumulation the topotecan-induced apoptosis, assessed according to DNA-fragmentation, chromatin condensation, and by determination of sub-G1 peaks using fluorescence-assisted-cell sorting (FACS), was potently inhibited by chrysin. Suggested transport-independent apoptosis inhibiting activities of ATP-binding cassette (ABC)-transporters, such as the inhibition of caspases, were shown to be necessary for the inhibition of topotecan-induced apoptosis and were found to be associated with stabilization of β-catenin especially in the cytosol. Inhibition of topotecan-induced intracellular acidification, however, was proven not to prevent caspase-activation and apoptosis.In conclusion, our studies show that chrysin in spite of raising the cellular concentrations of topotecan potently inhibits the apoptosis-inducing activities of the anti-tumor drug. Inhibition of caspase-activation was identified as the underlying mechanism and is suggested to be caused by transport-independent functions of ABC-transporters.

Keywords: Abbreviations; ABC; ATP-binding cassette; Ac-DEVD-CHO; acetyl-aspartyl-glutamyl-valyl-aspartyl-aldehyde; Ac-IETD-AFC; acetyl-isoleucyl-glutamyl-threonyl-aspartyl-7-amino-4-trifluoromethylcoumarin; AP; alkaline phosphatase; BCECF-AM; 2′,7′-; bis-; (2-carboxyethyl)-5-(and 6)-carboxyfluorescein acetoxymethylester; BCRP; breast cancer resistance protein; CMFDA; 5-chloromethylfluorescein diacetate; DMEM; Dulbecco's Modified Eagle Medium; ECL; enhanced chemiluminescence; DTT; dithiothreitol; FACS; fluorescence activated cell sorting; FCS; fetal calf serum; HEPES; N-hydroxyethylpiperazine-N′-2-ethanesulfonate; MDR; multi drug resistance; MRP; multidrug-resistance-associated protein; NEAA; nonessential amino acids; P-gp; P-phosphoglycoprotein; pH; _i; intracellular pH; TBS-T; Tris-buffered saline-Tween; Z-DEVD-AMC; carboxybenzoxy-aspartyl-glutamyl-valyl-aspartyl-amino-4-methyl-coumarineABC-transporters; Apoptosis; Caspases; Topotecan; Chrysin; β-Catenin

Small hyaluronan oligosaccharides induce inflammation by engaging both toll-like-4 and CD44 receptors in human chondrocytes by Giuseppe M. Campo; Angela Avenoso; Salvatore Campo; Angela D’Ascola; Giancarlo Nastasi; Alberto Calatroni (pp. 480-490).

Small hyaluronan (HA) oligosaccharides primed inflammation in normal human chondrocytes by stimulating both toll-like receptor 4 (TLR-4) and cluster determinant 44 (CD44) receptors. Blocking antibodies of these two receptors reduced nuclear factor kappaB (NF-κB) activation and inflammatory mediators.Small degradation fragments of hyaluronan (HA) may stimulate an inflammatory response in a variety of tissues at the injury site. HA oligosaccharides are endogenous ligands for the cluster determinant 44 (CD44) receptor as well as for toll-like receptor 4 (TLR-4). Previous data have shown that HA fragments may induce pro-inflammatory cytokine expression by interacting with both the CD44 receptor and TLR-4. CD44 and TLR-4 stimulation activates different inflammatory pathways that culminate with the activation of the transcriptional nuclear factor kappaB (NF-κB) which is responsible for the expression of inflammation mediators such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β). The aim of this study was to investigate the inflammatory effects of very small HA oligosaccharides on both TLR-4 and CD44 involvement in normal human articular chondrocytes.Adding HA fragments to chondrocyte cultures up-regulated CD44 and TLR-4 expression, activated NF-κB translocation and increased the pro-inflammatory cytokines TNF-α, IL-6 and IL-1β.The addition of a specific CD44 blocking antibody reduced CD44 and all inflammatory cytokine expression as well as protein production. However, cytokine expression remained significantly higher than in untreated chondrocytes. TLR-4 expression was not affected. The treatment with TLR-4 blocking antibody decreased TLR-4 and inflammatory cytokine expression, although cytokine expression was significantly higher than in control cells. CD44 expression was unaffected. The addition of both CD44 and TLR-4 blocking antibodies significantly reduced CD44, TLR-4 and inflammatory cytokine expression.

Keywords: Hyaluronan; Cytokines; Chondrocytes; NF-κB; TLR-4

Induction of indoleamine 2,3-dioxygenase expression via heme oxygenase-1-dependant pathway during murine dendritic cell maturation by In Duk Jung; Jun Sik Lee; Chang-Min Lee; Kyung Tae Noh; Yeong-Il Jeong; Won Sun Park; Sung Hak Chun; Soo Kyung Jeong; Jin Wook Park; Kwang Hee Son; Deok Rim Heo; Min-Goo Lee; Yong Kyoo Shin; Han Wool Kim; Cheol-Heui Yun; Yeong-Min Park (pp. 491-505).

HO-1-mediated IDO expression is dependent on the NF-κB pathway and constitutes an intermediate step in the DC maturation pathway.Heme oxygenase (HO)-1 is expressed in a variety of conditions involved in the regulation of immune responses. In this study, we examined the role of HO-1 in dendritic cell (DC) maturation and expression of indoleamine 2,3-dioxygenase (IDO), a key enzyme that catalyzes the initial, rate-limiting step in tryptophan degradation. IDO deficiency led to diminished phenotypic and functional maturation of DCs in vitro and in vivo. IDO expression and DC maturation was abrogated by the HO inhibitor zinc protoporphrin, but increased by hemin, a potent inducer of HO-1. Moreover, LPS-induced HO-1 expression was mediated by an NF-κB-dependent pathway. Our findings provide additional insight into the immunological functions of IDO and HO-1, and suggest possible therapeutic adjuvants for the treatment of DC-related acute and chronic diseases.

Keywords: Dendritic cells; Heme oxygenase; Indoleamine 2,3-dioxygenase; Lipopolysaccharide

Novel Alexa Fluor-488 labeled antagonist of the A_2A adenosine receptor: Application to a fluorescence polarization-based receptor binding assay by Miklós Kecskés; T. Santhosh Kumar; Lena Yoo; Zhan-Guo Gao; Kenneth A. Jacobson (pp. 506-511).

Fluorescence polarization (FP) assay has many advantages over the traditional radioreceptor binding studies. We developed an A_2A adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A_2AAR (MRS5346), a pyrazolo[4,3- e][1,2,4]triazolo[1,5- c]pyrimidin-5-amine derivative conjugated to the fluorescent dye Alexa Fluor-488. MRS5346 displayed a K_i value of 111±16nM in radioligand binding using [³H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A_2AAR. In a cyclic AMP functional assay, MRS5346 was shown to be an A_2AAR antagonist. MRS5346 did not show any effect on A₁ and A₃ ARs in binding or the A_2BAR in a cyclic AMP assay at 10μM. Its suitability as a fluorescent tracer was indicated in an initial observation of an FP signal following A_2AAR binding. The FP signal was optimal with 20nM MRS5346 and 150μg protein/mL HEK293 membranes. The association and dissociation kinetic parameters were readily determined using this FP assay. The K_d value of MRS5346 calculated from kinetic parameters was 16.5±4.7nM. In FP competition binding experiments using MRS5346 as a tracer, K_i values of known AR agonists and antagonists consistently agreed with K_i values from radioligand binding. Thus, this FP assay, which eliminates using radioisotopes, appears to be appropriate for both routine receptor binding and high-throughput screening with respect to speed of analysis, displaceable signal and precision. The approach used in the present study could be generally applicable to other GPCRs.

Keywords: Abbreviations; AF488; Alexa Fluor-488; CHO; Chinese hamster ovary; CCPA; 2-chloro-N; ⁶; -cyclopentyladenosine; CGS21680; 2-[; p; -(2-carboxyethyl)phenylethylamino]-5′-; N; -ethylcarboxamido-adenosine; DMEM; Dulbecco's Modified Eagle Medium; DMF; N; ,; N; -dimethylformamide; DMSO; dimethyl sulfoxide; EDTA; ethylenediaminetetraacetic acid; FBS; fetal bovine serum; FP; fluorescence polarization; GPCR; G protein-coupled receptor; HEK; human embryonic kidney; HTS; high-throughput screening; [; ¹²⁵; I]AB-MECA; [; ¹²⁵; I]4-amino-3-iodobenzyl-5′-; N; -methylcarboxamidoadenosine; mP; millipolarization; MRS5346; 5-((2-(2-(4-(3-(5-amino-2-(furan-2-yl)-7; H; -pyrazolo[4,3-; e; ][1,2,4]triazolo[1,5-; c; ]pyrimidin-7-yl)propyl)phenoxy)acetamido)ethyl)-carbamoyl)-2-(6-amino-3-iminio-4,5-disulfonato-3; H; -xanthen-9-yl)benzoate; NECA; 5′-; N; -ethylcarboxamidoadenosine; SCH442416; 2-(2-furyl)-7-[3-(4-methoxyphenyl)propyl]-7; H; -pyrazolo[4,3-; e; ][1,2,4]triazolo[1,5-; c; ]pyrimidin-5-amine; SCH58261; 2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine; XAC; xanthine amine congener, 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine; ZM241385; 4-[2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-yl-amino]ethylphenolPurines; Fluorescence polarization; G protein-coupled receptor; High-throughput screening; A; _2A; adenosine receptor

Green tea catechins are potent sensitizers of ryanodine receptor type 1 (RyR1) by Wei Feng; Gennady Cherednichenko; Chris W. Ward; Isela T. Padilla; Elaine Cabrales; José R. Lopez; José M. Eltit; Paul D. Allen; Isaac N. Pessah (pp. 512-521).

Catechins, polyphenols extracted from green tea leaves, have a broad range of biological activities although the specific molecular mechanisms responsible are not known. At the high experimental concentrations typically used polyphenols bind to membrane phospholipid and also are easily auto-oxidized to generate superoxide anion and semiquinones, and can adduct to protein thiols. We report that the type 1 ryanodine receptor (RyR1) is a molecular target that responds to nanomolar (−)-epigallocatechin-3-gallate (EGCG) and (−)-epicatechin-3-gallate (ECG). Single channel analyses demonstrate EGCG (5–10nM) increases channel open probability (Po) twofold, by lengthening open dwell time. The degree of channel activation is concentration-dependent and is rapidly and fully reversible. Four related catechins, EGCG, ECG, EGC ((−)-epigallocatechin) and EC ((−)-epicatechin) showed a rank order of activity toward RyR1 (EGCG>ECG≫EGC>>>EC). EGCG and ECG enhance the sensitivity of RyR1 to activation by ≤100μM cytoplasmic Ca²⁺ without altering inhibitory potency by >100μM Ca²⁺. EGCG as high as 10μM in the extracellular medium potentiated Ca²⁺ transient amplitudes evoked by electrical stimuli applied to intact myotubes and adult FDB fibers, without eliciting spontaneous Ca²⁺ release or slowing Ca²⁺ transient recovery. The results identify RyR1 as a sensitive target for the major tea catechins EGCG and ECG, and this interaction is likely to contribute to their observed biological activities.

Keywords: Abbreviations; EGCG; (−)-epigallocatechin-3-gallate; ECG; (−)-epicatechin-3-gallate; EGC; (−)-epigallocatechin; EC; (−)-epicatechin; E–C; excitation–contraction; SR; sarcoplasmic reticulum; RyR1; ryanodine receptor type 1; SERCA; sarcoplasmic/endoplasmic reticulum ATPaseGreen tea extracts; Polyphenols; Catechins; Ca; ²⁺; Ryanodine receptor; E–C coupling; Sarcoplasmic reticulum; Skeletal muscle

Characteristics of tolerance in the guinea pig ileum produced by chronic in vivo exposure to opioid versus cannabinoid agonists by Hercules Maguma; Kathleen Thayne; David A. Taylor (pp. 522-532).

Chronic opioid treatment induces heterologous tolerance by changing cell excitability while chronic cannabinoid treatment induces homologous tolerance by modifying the receptor population.Few studies have compared the nature of tolerance that develops following chronic opioid treatment with that which develops after chronic cannabinoid exposure in the same tissue and species. The degree and character of tolerance induced by 7 twice daily injections of morphine or 5 daily injections of the cannabinoid receptor agonist, WIN-55,212-2, was examined by comparing the ability of DAMGO, 2-chloroadenosine (CADO) and WIN-55,212-2 to inhibit neurogenic contractions of the longitudinal muscle/myenteric plexus preparation (LM/MP) and the ability of nicotine to elicit contractions in the LM/MP. Chronic morphine treatment resulted in subsensitivity to all inhibitory agonists (rightward shift in IC₅₀ values of 4–5-fold) and an increased responsiveness to the excitatory effect of nicotine while chronic WIN-55,212-2 exposure resulted in subsensitivity only to WIN-55,212-2 and a reduction in maximum response to both WIN-55,212-2 and DAMGO but no change in responsiveness to CADO. Chronic WIN-55,212-2 treatment significantly reduced CB₁ but not MOR receptor protein abundance while chronic morphine treatment did not change either. Assessment of the distribution of MOR and CB₁ receptors in myenteric neurons revealed distinct individual receptor expression as well as co-localization which was unaffected by either cannabinoid or opioid treatment. Thus, in contrast to the heterologous tolerance that develops after opioid treatment, tolerance in the LM/MP following chronic in vivo WIN-55,212-2 exposure appears to be homologous in character and is accompanied by a selective decrease in CB₁ receptor protein abundance. The data suggest that the cellular basis of tolerance differs between the two systems.

Keywords: Abbreviations; DAMGO; Tyr-; d; -Ala-Gly-N-methyl-Phe-Gly-ol; CADO; 2-chloroadenosine; LM/MP; longitudinal muscle/myenteric plexus; MOR; mu-opioid receptor; WIN-55,212-2; R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone; CB; ₁; cannabinoid receptor subtype 1Adaptation; Tolerance; Cannabinoid; Opioid; Guinea pig; Longitudinal muscle/myenteric plexus

The effect of dimerumic acid on LPS-induced downregulation of Mrp2 in the rat by Kentaro Yano; Shuichi Sekine; Kanako Nemoto; Toru Fuwa; Toshiharu Horie (pp. 533-539).

Oxidative stress is known to be a common feature of cholestatic syndrome. Lipopolysaccharide (LPS) induces cholestasis, causing multidrug resistance-associated protein 2 (Mrp2) downregulation in two different ways: early retrieval from the canalicular membrane and the latter event of reduced mRNA expression. However, the triggering factor for LPS-induced cholestasis is not fully understood.In this study, we examined the effect of dimerumic acid (DMA), an antioxidant and traditional Chinese medicine, on endotoxin-induced Mrp2 downregulation in rat liver. At 3h following LPS injection (4mg/kg body weight), canalicular Mrp2 localization was disrupted without changing the expression of Mrp2 protein or the integrity of tight junctions in the liver. Pretreatment with DMA (12mg/kg body weight) counteracted LPS-induced subcellular distribution, and decreased the bile flow rate and biliary glutathione (GSH) excretion. At 12h following LPS injection, Mrp2 protein and mRNA expression were significantly decreased by 58% and 7%, respectively. In contrast, pretreatment with DMA did not have any effect on the decreased Mrp2 expression and biliary excretion of GSH induced by LPS exposure. Taken together, our data clearly indicate that LPS-induced short-term rapid retrieval of Mrp2 from the canalicular surface resulted from LPS-induced oxidative stress, while the long-term transcriptional regulation of Mrp2 expression did not depend on the intracellular redox status.

Keywords: Abbreviations; Bsep; bile salt export pump; Mrp2; multidrug resistance-associated protein 2; GSH; glutathione; LPS; lipopolysaccharide; EA; ethacrynic acid; DMA; dimerumic acid; ALT; alanine aminotransferase; NO; nitric oxide; CrM; crude membrane; ZO-1; zonal occuldin-1; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; P-gp; P-glycoproteinDimerumic acid; Cholestasis; Internalization; Oxidative stress; Mrp2

A toxicoproteomic study on cardioprotective effects of pre-administration of docetaxel in a mouse model of adriamycin-induced cardiotoxicity by Kaname Ohyama; Mari Tomonari; Tomoko Ichibangase; Hideto To; Naoya Kishikawa; Kenichiro Nakashima; Kazuhiro Imai; Naotaka Kuroda (pp. 540-547).

Differential proteomic analysis of heart tissue by FD-LC–MS/MS.Studies suggest that pre-administration of docetaxel (DOC) in adriamycin (ADR)-DOC combination anticancer therapy results in stronger antitumor effects and fewer ADR-induced cardiotoxic deaths in mouse model, yet no mechanism explaining this effect has been established. The aim of this study was to identify cellular processes in mouse heart tissue affected by different ADR/DOC dosing protocols using a toxicoproteomic approach. We applied fluorogenic derivatization-liquid chromatography–tandem mass spectrometry (FD-LC–MS/MS) – which consists of fluorogenic derivatization, separation and fluorescence detection by LC, and identification by LC–tandem mass spectrometry – to the proteomic analysis of heart tissue from control, intermittent-dosing (DOC-ADR), and simultaneous-dosing (ADR&DOC) groups. In DOC-ADR group, ADR was administered 12h after DOC injection; in ADR&DOC group, both drugs were administered simultaneously; in control group, saline was administered at the same timing as ADR injection of other groups. Heart samples were isolated from all mice 1 week after the treatment. The highly reproducible and sensitive method (FD-LC–MS/MS) identified nine proteins that were differentially expressed in heart tissue of control and the two treatment groups; seven of these nine proteins participate in cellular energy production pathways, including glycolysis, the tricarboxylic acid cycle, and the mitochondrial electron transport chain. Significantly higher expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was observed in the DOC-ADR group, the group with the fewer cardiotoxic deaths, than in the ADR&DOC group. Therefore, GAPDH may have potential as a drug target for protective intervention and a biomarker for evaluation of the cardioprotective effects in pre-clinical studies.

Keywords: Adriamycin-induced cardiotoxicity; Docetaxel pre-administration; Fluorogenic derivatization-liquid chromatography–tandem mass spectrometry; Toxicoproteomics

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Biochemical Pharmacology (v.80, #4)