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Biochemical Pharmacology (v.70, #5)

Editorial Advisory Board (pp. iii).

Controlling cytokine signaling by constitutive inhibitors by Kriti Rakesh; Devendra K. Agrawal (pp. 649-657).

Cytokines are secreted proteins that regulate diverse biological functions by binding to receptors at the cell surface to activate complex signal transduction pathways including the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Stringent mechanisms of signal attenuation are essential for ensuring an appropriate, controlled cellular response. Three families of proteins, the SH2-containing phosphatases (SHP), the protein inhibitors of activated STATs (PIAS), and the suppressors of cytokine signaling (SOCS), inhibit specific and distinct aspects of cytokine signal transduction. The analysis of mice lacking genes for members of the SHP has shed much light on the roles of these proteins in vivo. In recent in vitro studies, the protein modifiers ubiquitin and small ubiquitin-like modifier (SUMO) have emerged as key players in the strategies employed by SOCS and PIAS to repress signaling. This review throws light on the mechanisms of action of these regulators as being evolved by the latest researches.

Keywords: Cytokines; JAK/STAT signalling; SOCS; SHP; PIAS; Inhibitors of signaling

Combination of cyclooxygenase-2 inhibitors and oxaliplatin increases the growth inhibition and death in human colon cancer cells by Johnson Lin; Po-Wen Hsiao; Ted H. Chiu; Jui-I Chao (pp. 658-667).

The cyclooxygenase-2 (COX-2) protein is highly expressed in a variety of human cancers and has been reported to promote tumor growth. Non-steroidal anti-inflammatory drugs such as etodolac and celecoxib have been shown to inhibit COX-2 activity and may play a role in the chemoprevention of cancer. Oxaliplatin is a third-generation platinum compound that exhibits a different spectrum of activity compared with cisplatin. Other cisplatin-resistant tumors can still respond to oxaliplatin. However, the anticancer ability of the combination of COX-2 inhibitors and oxaliplatin is still unknown. In this study, we investigated the effects of combination of COX-2 inhibitors and oxaliplatin on the cell growth and survival in human colon cancer cells. Treatments with etodolac (0.3–0.5mM) or celecoxib (20–80μM) for 24h concentration-dependently induced the cytotoxicity in the RKO colon carcinoma cells. Etodolac and celecoxib did not alter the COX-2 protein levels but inhibited its enzyme activity to reduce prostaglandin E₂ production. Furthermore, the cell survival was concentration-dependently decreased following oxaliplatin (1–100μM, 24h) treatment. Combination of oxaliplatin and etodolac additively increased the death and growth inhibition of RKO cells. Survivin, an inhibitor protein of apoptosis, mediates anti-apoptosis and promotes cell division in cancer cells. Oxaliplatin or COX-2 inhibitors significantly decreased the levels of survivin proteins. Moreover, survivin proteins were markedly diminished following co-treatment with oxaliplatin and etodolac. Together, this is the first report that combination of COX-2 inhibitors and oxaliplatin can increase the reduction of survivin protein expression, growth inhibition, and death in human colon cancer cells.

Keywords: Abbreviations; COX-2; cyclooxygenase-2; NSAID; non-steroidal anti-inflammatory drug; MTT; 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; FITC; fluorescein isothiocyanate; FBS; fetal bovine serum; PBS; phosphate-buffered saline; ERK; extracellular signal-regulated kinase; F-actin; actin filament; PG; prostaglandinColon cancer; Celecoxib; COX-2; Etodolac; Oxalipatin; Survivin

An effect of antibiotic amphotericin B on ion transport across model lipid membranes and tonoplast membranes by Monika Hereć; Halina Dziubińska; Kazimierz Trębacz; Jacek W. Morzycki; Wiesław I. Gruszecki (pp. 668-675).

A pH sensitive fluorescence probe piranine trisulfonate, entrapped inside small unilamellar liposomes formed with egg yolk phosphatidylcholine, was applied to investigate effect of polyene antibiotic amphotericin B (AmB) on proton transport across lipid membranes. Time dependencies of fluorescence-monitored pH changes inside lipid vesicles, upon sudden acidification of the liposome suspension, were analyzed in terms of two-exponential kinetics. It appears that addition of AmB at 3mol%, with respect to lipid, considerably increases the rate constant of the fast component of proton transport (a change from (60 to 149)×10⁻³s⁻¹) and decreases the rate constant of the slow component (a change from (11 to 5)×10⁻³s⁻¹). Incorporation of 0.1mol% AmB results in the decrease of both parameters (to (33 and 2)×10⁻³s⁻¹, respectively). The increase in the rate of proton transfer across the lipid membrane is interpreted as related to the formation of membrane channels by AmB, at higher concentration of the drug or nonspecific destabilization of the membrane structure. At low concentrations, at which formation of molecular structures of AmB is not possible, the antibiotic molecules are oriented horizontally with respect to the plane of the membrane and act in making the membrane more compact and less permeable to ions. The presence of sterols (cholesterol, ergosterol and cholesterol dimer) in the lipid phase, in the concentration 3mol% and lower, decreased the rate constants of proton transfer across the membranes but did not influence significantly the effect of AmB on the ion transport. The presence of AmB in the bathing solutions of tonoplast membranes isolated from Conocephalum conicum at the concentrations range 1×10⁻⁷ to 3.6×10⁻⁵ does not influence considerably the ion current, as monitored by means of the patch-clamp technique.

Keywords: Amphotericin B; Liposomes; Proton transport; Polyene antibiotics; Patch-clamp

Structural requirements of Dictyostelium differentiation-inducing factors for their stalk-cell-inducing activity in Dictyostelium cells and anti-proliferative activity in K562 human leukemic cells by Naomi Gokan; Haruhisa Kikuchi; Koji Nakamura; Yoshiteru Oshima; Kohei Hosaka; Yuzuru Kubohara (pp. 676-685).

The differentiation-inducing factor-1 (DIF-1) is a lipophilic signal molecule (chlorinated alkylphenone) that induces stalk-cell differentiation in the cellular slime mould Dictyostelium discoideum. It has also been shown that DIF-1 and its derivative (DIF-3) suppress cell growth in mammalian tumor cells. In the present study, in order to assess the chemical structure–effect relationship of DIF derivatives and to develop useful agents for the study of both Dictyostelium development and cancer biology, we synthesized 28 analogues of DIF-1 and DIF-3 and investigated their stalk-cell-inducing activity in Dictyostelium HM44 cells (mutant strain) and anti-proliferative activity in human leukemia K562 cells. HM44 cells are defective in endogenous DIF-1 production and should be suitable for the assay for stalk-cell-inducing activity of DIF analogues. DIF-1 and some of its derivatives at nanomolar levels were good stalk-cell inducers in HM44 cells, whereas DIF-3 and some DIF-3 derivatives at micromolar levels were potent anti-proliferative agents in K562 cells. We also tried to search for antagonistic molecules against DIF-1 and DIF-3 but failed to find such molecules from the analogues used here. The present findings would give us hints for identifying the target molecule(s) of DIFs and also for developing novel anti-cancer drugs.

Keywords: Abbreviations; [Ca; ²⁺; ]; _i; intracellular calcium concentration; DIF-1; differentiation-inducing factor-1; DIF-3; differentiation-inducing factor-3 Dictyostelium; K562; DIF-1; DIF-3; Stalk-cell differentiation; Anti-cancer drug

Differential regulation of NPR-B/GC-B by protein kinase c and calcium by Sarah E. Abbey-Hosch; Dmitri Smirnov; Lincoln R. Potter (pp. 686-694).

C-type natriuretic peptide (CNP) activation of the guanylyl cyclase-linked natriuretic peptide receptor-B (NPR-B) stimulates vasorelaxation and bone growth. Hormones and phorbol esters (PMA) inhibit NPR-B in calcium and protein kinase c-dependent manners, respectively. Here, we characterize the kinetic properties of NPR-B in membranes from cells exposed to PMA, the calcium ionophore, ionomycin, or sphingosine-1-phosphate (S1P). PMA and ionomycin primarily increased the K_m and decreased the V_max of NPR-B for GTP, respectively, whereas S1P caused modest changes in both parameters. PMA and S1P treatment increased the EC₅₀ for CNP activation by eight- and three-fold, whereas ionomycin was ineffective. All three agents caused NPR-B dephosphorylation, but the basis for the loss of phosphate differed between treatments. In vitro phosphorylation of NPR-B in membranes was markedly diminished by prior whole cell PMA or S1P exposure, whereas ionomycin pretreatment had no effect. The involvement of the known phosphorylated residues in each process was tested with a mutant receptor containing glutamates substituted for these sites. While the effect of PMA was lost on this receptor, the effects of S1P and ionomycin were only partially blocked. Our data suggest that the molecular bases for PMA- and calcium-dependent inhibition of NPR-B are unique. The former results from reduced phosphorylation of a known site and primarily affects the affinity of NPR-B for CNP and GTP. The latter is associated with reductions in maximal velocities by a mechanism that does not involve inhibition of NPR-B phosphorylation and requires a process in addition to the dephosphorylation of the known sites.

Keywords: Abbreviations; ANP; atrial natriuretic peptide; BNP; b-type natriuretic peptide; CNP; C-type natriuretic peptide; KHD; kinase homology domain; AVP; arginine-vasopressin; NPR-A; natriuretic peptide receptor A; NPR-B; natriuretic peptide receptor B; S1P; sphingosine-1-phosphate; PMA; phorbol 12-myristate 13-acetateANF; NPR-B; Guanylyl cyclase; cGMP; PKG; GC-A

Distribution of breast cancer resistance protein (BCRP/ABCG2) mRNA expression along the human GI tract by Heike Gutmann; Petr Hruz; Christian Zimmermann; Christoph Beglinger; Juergen Drewe (pp. 695-699).

Human breast cancer resistance protein (BCRP/ABCG2) is an ABC-transporter that is present on the luminal membrane of intestinal epithelial cells and restricts absorption of anticancer drugs such as methotrexate, topotecan, mitoxantrone, and doxorubicin. The exact anatomic distribution of BCRP along the gastrointestinal (GI) tract, however, has not been determined before. The aim of this study was, therefore to investigate BCRP mRNA expression pattern along the GI tract in 14 healthy subjects. Furthermore, BCRP duodenal mRNA expression was compared with MDR1/ABCB1 mRNA. Additionally, BCRP mRNA expression was investigated in two human intestinal cell lines (Caco-2 and LS180). Since previous animal studies have suggested sex specific differences in BCRP expression, we analyzed intestinal BCRP expression with respect to sex. Biopsies were taken from different gut segments (duodenum, terminal ileum and ascending, transverse, descending and sigmoid colon). Gene expression was assessed by quantitative real-time PCR (Taqman). BCRP mRNA expression was maximal in the duodenum and decreased continuously down to the rectum (terminal ileum 93.7%, ascending colon 75.8%, transverse colon 66.6%, descending colon 62.8%, and sigmoid colon 50.1% compared to duodenum, respectively). BCRP expression in the duodenum was comparable to MDR1/ABCB1 gene expression. Caco-2 cells showed a comparable expression of BCRP as human duodenal tissue. Gender specific differences in BCRP expression were not observed. These findings represent the first systematic site-specific analysis of BCRP expression along the GI tract. This information might be helpful to develop target strategies for orally administered anticancer drugs.

Keywords: Abbreviations; BCRP; human breast cancer resistance protein; ABCG2; human breast cancer resistance protein; PCR; polymerase chain reactionBCRP; ABCG2; MDR1; ABCB1; Intestinal mapping; Human; Caco-2 cells; LS180 cells

Curcumin (diferuloylmethane) inhibits constitutive NF-κB activation, induces G1/S arrest, suppresses proliferation, and induces apoptosis in mantle cell lymphoma by Shishir Shishodia; Hesham M. Amin; Raymond Lai; Bharat B. Aggarwal (pp. 700-713).

Human mantle cell lymphoma (MCL), an aggressive B cell non-Hodgkin's lymphoma, is characterized by the overexpression of cyclin D1 which plays an essential role in the survival and proliferation of MCL. Because of MCL's resistance to current chemotherapy, novel approaches are needed. Since MCL cells are known to overexpress NF-κB regulated gene products (including cyclin D1), we used curcumin, a pharmacologically safe agent, to target NF-κB in a variety of MCL cell lines. All four MCL cell lines examined had overexpression of cyclin D1, constitutive active NF-κB and IκB kinase and phosphorylated forms of IκBα and p65. This correlated with expression of TNF, IκBα, Bcl-2, Bcl-xl, COX-2 and IL-6, all regulated by NF-κB. On treatment of cells with curcumin, however, downregulated constitutive active NF-κB and inhibited the consitutively active IκBα kinase (IKK), and phosphorylation of IκBα and p65. Curcumin also inhibited constitutive activation of Akt, needed for IKK activation. Consequently, the expression of all NF-κB-regulated gene products, were downregulated by the polyphenol leading to the suppression of proliferation, cell cycle arrest at the G1/S phase of the cell cycle and induction of apoptosis as indicated by caspase activation, PARP cleavage, and annexin V staining. That NF-κB activation is directly linked to the proliferation of cells, is also indicated by the observation that peptide derived from the IKK/NEMO-binding domain and p65 suppressed the constitutive active NF-κB complex and inhibited the proliferation of MCL cells. Constitutive NF-κB activation was found to be due to TNF, as anti-TNF antibodies inhibited both NF-κB activation and proliferation of cells. Overall, our results indicate that curcumin inhibits the constitutive NF-κB and IKK leading to suppression of expression of NF-κB-regulated gene products that results in the suppression of proliferation, cell cycle arrest, and induction of apoptosis in MCL.

Keywords: Abbreviations; EMSA; electrophoretic mobility shift assay; IKK; IκB kinase; FBS; fetal bovine serum; IκBα; inhibitory subunit of NF-κB; MCL; mantle cell lymphoma; NF-κB; nuclear transcription factor-κB; NEMO; NF-κB essential modifier; NBD; NEMO-binding domain peptide; PI; propidium iodide; PIS; pre-immune serum; HRP; horse radish peroxidase; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PMSF; phenylmethylsulfonyl fluoride; EBV; Epstein-Barr virus; PTD; protein transduction domainMCL; NF-κB; IκBα; IKK; Curcumin

Prostaglandin E₂ induces the expression of functional inhibitory CD94/NKG2A receptors in human CD8⁺ T lymphocytes by a cAMP-dependent protein kinase A type I pathway by Mustapha Zeddou; Roland Greimers; Nicolas de Valensart; Btissam Nayjib; Kjetil Tasken; Jacques Boniver; Michel Moutschen; Souad Rahmouni (pp. 714-724).

The CD94/NKG2A heterodimer is a natural killer receptor (NKR), which inhibits cell-mediated cytotoxicity upon interaction with MHC class I gene products. It is expressed by NK cells and by a small fraction of activated T cells, predominantly of CD8⁺ phenotype. Abnormal upregulation of the CD94/NKG2A inhibitory NKR on cytotoxic T cells (CTLs) could be responsible for a failure of immunosurveillance in cancer or HIV infection. In an attempt to identify the mechanisms leading to inhibitory NKR upregulation on T cells, we analyzed the expression of the CD94/NKG2A heterodimer on human CTLs activated with anti-CD3 mAb in the presence of PGE₂ or with 8-CPT-cAMP, an analogue of cyclic AMP. As previously described, anti-CD3 mAb-mediated activation induced the expression of CD94/NKG2A on a small fraction of CD8⁺ T cells. Interestingly, when low concentrations of PGE₂ or 8-CPT-cAMP were present during the culture, the proportion of CD8⁺ T cells expressing CD94/NKG2A was two- to five-fold higher. This upregulation was partially prevented by PKA inhibitors, such as KT5720 and Rp-8-Br-cAMP (type I selective). We also report that cAMP induces upregulation of NKG2A at the mRNA level. We further demonstrated that cross-linking of CD94 on CD8⁺ T cells expressing the CD94/NKG2A heterodimer inhibits their cytotoxic activity in a bispecific antibody redirected lysis assay. Our findings clearly demonstrate that the PGE₂/cAMP/PKA type I axis is involved in the expression of CD94/NKG2A receptor on human CD8⁺ T lymphocytes.

Keywords: Human; T lymphocytes; Cell surface molecules; Cytotoxicity

Modulation of epirubicin cytotoxicity by tamoxifen in human breast cancer cell lines by Samar S. Azab; Ebtehal El-Demerdash; Ashraf B. Abdel-Naim; Ekram Youssef; Nahla El-Sharkawy; Abdel-Moneim M. Osman (pp. 725-732).

The present study was designed to investigate the modulatory effect of the anti-estrogen, tamoxifen (Tam) on epirubicin (Epi) cytotoxicity in breast cancer cell lines; MCF-7 and NCI-adr. Using sulphorhodamine-B assay, NCI-adr cell line was found to be five-folds more resistant to the cytotoxic effect of Epi as compared to MCF-7 cell line. Pretreatment of cells with Tam was observed to enhance Epi cytotoxicity by 4.3- and 6.5-folds in MCF-7 and NCI-adr cells, respectively. Tam–Epi interaction was found to be additive in MCF-7 cells and synergistic in NCI-adr cells. Flowcytometric DNA ploidy analysis revealed that, Epi induced cell arrest at G₂/M phase. Tam pretreatment enhanced the blocking activity of low dose of Epi in MCF-7 and induced nearly two-fold increase in the percentage of S phase in NCI-adr cells. Determination of cellular Epi level revealed that Tam induced a significant increase in intracellular Epi accumulation only in NCI-adr cells after 48h. However, analysis of P-gp function revealed that Tam failed to modulate P-gp function in both cell lines. Also, assessment of topoisomerae IIα gene expression showed that neither Epi nor Tam managed to change its expression level. In conclusion, Tam potentiates Epi cytotoxicity in sensitive and resistant breast cancer cell lines. This potentiation can be explained by an enhancement of cell accumulation in S and G₂/M phase, at which the cells are most sensitive to the cytotoxic effect of Epi as well as an increase in the intracellular level of Epi in resistant cell line.

Keywords: Epirubicin; Multi-drug resistance; Tamoxifen; Cell cycle; P-glycoprotein; Topoisomerase IIα

Role of Ca²⁺-independent phospholipase A₂ and cytochrome P-450 in store-operated calcium entry in 3T6 fibroblasts by Javier Mart�nez; Juan J. Moreno (pp. 733-739).

Store-operated calcium (SOC) channels and capacitative Ca²⁺ entry play a key role in cellular functions, but their mechanism of activation remains unclear. Here, we show that thapsigargin induces [³H] arachidonic acid (AA) release,⁴⁵Ca²⁺ influx and a subsequent enhancement of intracellular calcium concentration ([Ca²⁺]_i. Thapsigargin-induced elevation of [Ca²⁺]_i was inhibited by cytochrome P-450 inhibitors and by cytochrome P-450 epoxygenase inhibitor and was reverted by 11,12 EET addition. However, cyclooxygenase and lipoxygenase inhibitors have no effect. Moreover, we observed that four EETs were able to induce⁴⁵Ca²⁺ influx. Finally, we reported that the effect of 11,12 EET on⁴⁵Ca²⁺ influx was sensible to receptor-operated Ca²⁺ channel blockers (NiCl₂, LaCl₃) but not to voltage-dependent Ca²⁺ channel blocker as verapamil. Thus, AA released by Ca²⁺-independent phospholipase A₂ and AA metabolism through cytochrome P-450 pathway may be crucial molecular determinant in thapsigargin activation of SOC channels and store-operated Ca²⁺ entry pathway in 3T6 fibroblasts. Moreover, EETs, the main cytochrome P-450 epoxygenase metabolites of AA, are involved in thapsigargin-stimulated Ca²⁺ influx. In summary, our results suggest that EETs are components of calcium influx factor(s).

Keywords: Abbreviations; AA; arachidonic acid; AACOCF; ₃; arachidonyl trifluoromethylketone; ARC; arachidonate-regulated Ca; ²⁺; BEL; bromoenol lactone; CCE; capacitative calcium entry; CRAC; calcium release-activated Ca; ²⁺; channels; CIF; calcium influx factor; EET; epoxyeicosatrienoic acid; ER; endoplasmic reticulum; FCS; fetal calf serum; InsP; ₃; inositol trisphosphates; [Ca; ²⁺; ]; _i; intracellular calcium concentrations; iPLA; ₂; calcium-independent phospholipase A; ₂; PLA; ₂; phospholipase A; ₂; PPOH; 6-(2 propargyloxyphenyl) hexanoic acid; SOC; store-operated calciumCalcium channels; Capacitative calcium entry; Epoxyeicosatrienoic acids; Arachidonic acid; Eicosanoids

Protective effects of Ca²⁺ handling drugs against abnormal Ca²⁺ homeostasis and cell damage in myopathic skeletal muscle cells by Yuko Iwata; Yuki Katanosaka; Zhu Shijun; Yuko Kobayashi; Hironori Hanada; Munekazu Shigekawa; Shigeo Wakabayashi (pp. 740-751).

Deficiency of δ-sarcoglycan (δ-SG), a component of the dystrophin–glycoprotein complex (DGC), causes skeletal muscular dystrophy and cardiomyopathy in BIO14.6 hamsters. Here, we studied the involvement of abnormal Ca²⁺ homeostasis in muscle degeneration and the protective effect of drugs against Ca²⁺ handling proteins in vivo as well as in vitro. First, we characterized the properties of cultured myotubes from muscles of normal and BIO14.6 hamsters (30–60 days old). While there were no apparent differences in the levels of expression of various Ca²⁺ handling proteins (L-type Ca²⁺ channel, ryanodine receptor, SR-Ca²⁺ ATPase, and Na⁺/Ca²⁺ exchanger), muscle-specific proteins (contractile actin and acetylcholine receptor), or DGC member proteins except SGs, BIO14.6 myotubes showed a high degree of susceptibility to mechanical stressors, such as cyclic stretching and hypo-osmotic stress as compared to normal myotubes, as evidenced by marked increases in creatine phosphokinase (CK) release and bleb formation. BIO14.6 myotubes showed abnormal Ca²⁺ homeostasis characterized by elevated cytosolic Ca²⁺ concentration, frequent Ca²⁺ oscillation, and increased⁴⁵Ca²⁺ uptake. These abnormal Ca²⁺ events and CK release were significantly prevented by Ca²⁺ handling drugs, tranilast, diltiazem, and FK506. The calpain inhibitor E64 prevented CK release, but not⁴⁵Ca²⁺ uptake. Some of these drugs (tranilast, diltiazem, and FK506) also exerted a significant protective effect for muscle degeneration in BIO14.6 hamsters and mdx mice in vivo. These observations suggest that elevated Ca²⁺ entry through sarcolemmal Ca²⁺ channels predominantly contributes to muscle degeneration and that the drugs tested here may have novel therapeutic potential against muscular dystrophy.

Keywords: Muscular dystrophy; Mechanical stretch; Ca; ²⁺; homeostasis; Ca; ²⁺; -permeable channel; Ca; ²⁺; influx; Cell damage

AMD3465, a monomacrocyclic CXCR4 antagonist and potent HIV entry inhibitor by Sigrid Hatse; Katrien Princen; Erik De Clercq; Mette M. Rosenkilde; Thue W. Schwartz; Pedro E. Hernandez-Abad; Renato T. Skerlj; Gary J. Bridger; Dominique Schols (pp. 752-761).

The chemokine receptors CCR5 and CXCR4 function as coreceptors for human immunodeficiency virus (HIV) and are attractive targets for the development of anti-HIV drugs. The most potent CXCR4 antagonists described until today are the bicyclams. The prototype compound, AMD3100, exhibits potent and selective anti-HIV activity against CXCR4-using (X4) viruses and showed antiviral efficacy in X4 HIV-1-infected persons in a phase II clinical trial. However, AMD3100 lacks oral bioavailability due to its high overall positive charge. Initial structure-activity relationship studies with bicyclam analogues suggested that the bis-macrocyclic structure was a prerequisite for anti-HIV activity. Now, we report that the N-pyridinylmethylene cyclam AMD3465, which lacks the structural constraints mentioned above, fully conserves all the biological properties of AMD3100. Like AMD3100, AMD3465 blocked the cell surface binding of both CXCL12 (the natural CXCR4 ligand), and the specific anti-CXCR4 monoclonal antibody 12G5. AMD3465 dose-dependently inhibited intracellular calcium signaling, chemotaxis, CXCR4 endocytosis and mitogen-activated protein kinase phosphorylation induced by CXCL12. Compared to the bicyclam AMD3100, AMD3465 was even 10-fold more effective as a CXCR4 antagonist, while showing no interaction whatsoever with CCR5. As expected, AMD3465 proved highly potent against X4 HIV strains (IC₅₀: 1–10nM), but completely failed to inhibit the replication of CCR5-using (R5) viruses. In conclusion, AMD3465 is a novel, monomacrocyclic anti-HIV agent that specifically blocks the interaction of HIV gp120 with CXCR4. Although oral bioavailability is not yet achieved, the monocyclams, with their decreased molecular charge as compared to the bicyclams, embody an important step forward in the design of oral CXCR4 antagonists that can be clinically used as anti-HIV drugs.

Keywords: Abbreviations; HIV; human immunodeficiency virus; CCR5; CC-chemokine receptor 5; CXCR4; CXC-chemokine receptor 4; AIDS; acquired immune deficiency syndrome; IC; ₅₀; 50% inhibitory concentration; CXCL12; CXC-chemokine ligand 12 (formerly ‘stromal cell-derived factor-1’); CXCL12; ^AF647; alexa fluor 647-conjugated CXCL12; PE; phycoerythrin; (m)Ab; (monoclonal) antibody; MAPK; mitogen-activated protein kinase; FBS; fetal bovine serum; GFP; green fluorescent protein; PBMC; peripheral blood mononuclear cell; IL-2; interleukin-2; PHA; phytohemagglutinin; MFI; mean fluorescence intensity; MTS; (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; ELISA; enzyme-linked immuno-sorbent assay; CC; ₅₀; 50% cytotoxic concentrationHIV entry; CXCR4 antagonist; Chemokine binding; Calcium signaling; MAP kinase; Chemotaxis

The effects of desmethylimipramine on cyclic AMP-stimulated gene transcription in a model cell system by J.K. Richards; W. Abdel-Razaq; T.E. Bates; D.A. Kendall (pp. 762-769).

The present study utilised an in vitro cell model of the cAMP signalling pathway to investigate the actions of desipramine (DMI) and other psychoactive agents on cAMP-driven gene transcription. The model comprised CHOβ₂ SPAP cells; Chinese hamster ovary cells expressing human β₂ adrenoceptors and a secreted placental alkaline phosphatase (SPAP) reporter gene with multiple cAMP response elements (CREs) in its promoter region. SPAP assays showed DMI to inhibit isoprenaline or forskolin-enhanced gene transcription in a time and concentration-dependent manner (IC₅₀=16.6±2.0μM after 18h). This effect of DMI was not dependent upon activity at the levels of the β₂ receptor, cAMP accumulation or phosphorylation of the transcription factor, cAMP response element binding protein (CREB). The inhibitory effects were maintained in the presence of DMI for at least 3 weeks and were mimicked by exposure to norfluoxetine (the major metabolite of fluoxetine; IC₅₀=7.2±1.8μM) and the neuroleptics, chlorpromazine and clozapine, all at a concentration of 10μM. Amphetamine (10μM, 18h) enhanced SPAP gene transcription. Ca²⁺ imaging experiments ruled out an inhibitory effect of DMI on Ca²⁺ influx as concluded by previous studies.The results suggest a molecular target for DMI that lies downstream of CREB phosphorylation. Whether the inhibitory action of DMI is common to naturally expressed CRE-driven genes involved in adaptive responses to antidepressants in vivo remains to be determined.

Keywords: Antidepressant; Cyclic AMP; CHO cell; CREB

Presence of diverse functional P2X receptors in rat cerebellar synaptic terminals by Cristina Herv�s; Raquel P�rez-Sen; M Teresa Miras-Portugal (pp. 770-785).

Studies in individual synaptic terminals have demonstrated the presence of diverse functional P2X receptors in rat cerebellum. No immunolabelling for P2X₁, P2X₄, P2X₅ and P2X₆, and scarce presence of P2X₂ were found at the cerebellar synaptic terminals. P2X₃ immunolabelling was present in 28% of isolated synaptosomes. At these synaptic terminals, nucleotides as ATP or α,β-meATP induced Ca²⁺ transients in the presence of extracellular Ca²⁺, showing homologous and heterologous receptor desensitization in 60% of cases. Ip₅I 10nM did not block responses to α,β-meATP, but inhibition occurred when antagonist concentrations were equal or higher than 100nM. These data agree with the presence of abundant P2X₃ homomeric receptors. P2X₇ immunolabelling was present in 60% of terminals and P2X₇ receptor hallmarks in Ca²⁺ responses have been found. BzATP was more potent than ATP and responses were potentiated when assayed in Mg²⁺-free medium. EC₅₀ values were, respectively, 39.4±0.4 and 0.3±0.1μM for ATP in the presence or absence of Mg²⁺. Maximal values of synaptosomal calcium transients, in the presence or absence of Mg²⁺, were, respectively, 91.6±11.9 and 132.9±12.9nM for ATP; and 104.3±9.4 and 169.7±17.1nM for BzATP. In addition, Zn²⁺ inhibited ATP responses in the absence of Mg²⁺ and the P2X₇ specific antagonist Brilliant Blue G completely blocked these responses in one half of synaptosomes. This study reports the presence of functional P2X₃ and P2X₇ receptors at synaptic sites, which provides complexity and regulatory possibilities to the cerebellar neurotransmission.

Non-stimulated Ca²⁺ leak pathway in cerebellar granule neurones by P.J. G�mez Pinilla; A.T. Hern�ndez; M.C. Camello; M.J. Pozo; E.C. Toescu; P.J. Camello (pp. 786-793).

The aim of this study was to investigate the pathways of calcium influx routes in non-stimulated cerebellar granule neurones by use of standard microspectrofluorimetric techniques. Repetitive application of Ca²⁺-free solutions for various time intervals induced decreases of resting cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) which were followed, on Ca²⁺ readmission, by a full recovery, always to the initial resting [Ca²⁺]_i levels. Use of drugs to deplete calcium stores (thapsigargin, alone or combined with low levels of ionomycin) did not cause release of Ca²⁺ from the intracellular stores nor enhanced the activity of the Ca²⁺ entry pathway. This influx was mainly independent of voltage operated calcium channels, since both L-type channel blockers (nitrendipine) and the hyperpolarizing agent pinacidil (a K⁺-channel opener) were without effect. Contribution from glutamate receptors to this influx was eliminated since a combination of blockers of NMDA and AMPA glutamate receptors (NBQX and D-AP5) did not affect the properties of the Ca²⁺ response. The Ca²⁺ leak pathway was sensitive to micromolar levels of lanthanum and gadolinium, and to the compound 2-APB, features shared by several channels of the TRP superfamily. In summary, our results show the presence of a Ca²⁺ permeable pathway, active and patent in resting conditions in cerebellar granule neurones, and which is different from the voltage-operated calcium channels and not operated by depletion of the stores.

Keywords: Cerebellar granule neurones; Calcium signal; Calcium entry; Calcium leak

Isolation and pharmacological characterisation of papuantoxin-1, a postsynaptic neurotoxin from the venom of the Papuan black snake ( Pseudechis papuanus) by Sanjaya Kuruppu; Shane Reeve; A. Ian Smith; Wayne C. Hodgson (pp. 794-800).

The Papuan black snake ( Pseudechis papuanus) is found throughout the southern coastal regions of Papua New Guinea and is thought to occur in the adjacent region of Iriyan Jaya. Neurotoxicity is a major symptom of envenomation by this species. This study describes the isolation of the first neurotoxin papuantoxin-1 from the venom of P. papuanus. Papuantoxin-1 (6738Da), which accounts for approximately 5% of the whole venom, was purified to homogeneity using successive steps of RP-HPLC. The toxin (0.3–1.0μM) caused concentration dependent inhibition of indirect twitches (0.1Hz, 0.2ms and supramaximal V) and inhibited the responses to nicotinic agonists in the chick biventer cervicis nerve-muscle preparation, indicating a postsynaptic mode of action. However, papuantoxin-1 displayed no signs of myotoxicity. Papuantoxin-1 displayed pseudo-irreversible antagonism of cumulative concentration-response curves to carbachol at the skeletal muscle nicotinic receptors with an estimated pA₂ value of 6.9±0.3. CSL black snake antivenom, which is raised against the venom of the Australian black snake Pseudechis australis, appears to be effective in reversing the effects of papuantoxin-1. Thus, black snake antivenom should be considered for the treatment of the neurotoxic effects following envenomation by the Papaun black snake.

Keywords: Neurotoxin; Snake; Venom; Antivenom; Skeletal muscle; Chick biventer

A novel CYP2A6*20 allele found in African-American population produces a truncated protein lacking enzymatic activity by Tatsuki Fukami; Miki Nakajima; Eriko Higashi; Hiroyuki Yamanaka; Howard L. McLeod; Tsuyoshi Yokoi (pp. 801-808).

Human CYP2A6 is a cytochrome P450 (CYP) isoform responsible for the metabolism of nicotine, coumarin, tegafur, and valproic acid, and metabolic activation of nitrosamines. Genetic polymorphisms of the CYP2A6 gene are a major causal factor of the large interindividual differences in nicotine metabolism. In the present study, we identified a novel allele, termed CYP2A6*20, in an African-American population. The allele possesses the deletion of two nucleotides in exon 4 resulting in a frame-shift from codon 196 and an early stop codon at 220 (exon 5) as well as three synonymous SNPs of G51A (G51A in cDNA), T5684C (T1191C), and C6692G (C1546G, 3′-untranslated region). The allele frequency in the African-American population ( n=96) was 1.6% (95% confidence interval, 0.6–4.5%). In contrast, the CYP2A6*20 allele was not found in Caucasians (European-American) ( n=185), Japanese ( n=184) and Korean ( n=209) populations. To investigate the effects of the polymorphism on the enzymatic activities, we expressed a wild type or variant (deletion of two nucleotides) CYP2A6 together with NADPH-CYP reductase in Escherichia coli. SDS-PAGE and immunoblot analyses demonstrated that truncated CYP2A6 protein was produced from the variant allele, although detected mRNA was the predicted size by reverse transcriptional-polymerase chain reaction. Coumarin 7-hydroxylation and nicotine C-oxidation, which are typical CYP2A6 activities, were completely abolished in the E. coli membrane expressing the variant allele. In vivo nicotine metabolism was evaluated using the cotinine/nicotine ratio 2h after the chewing of one piece of nicotine gum. Two CYP2A6*1/ CYP2A6*20 heterozygotes and a single CYP2A6*17/ CYP2A6*20 heterozygote revealed lower cotinine/nicotine ratios compared with CYP2A6*1/ CYP2A6*1 subjects (1.6 and 4.5, and 1.8 versus 9.5±5.4, n=52, respectively). We found a novel CYP2A6*20 allele in African-American subjects which codes a truncated protein lacking enzymatic activity.

Keywords: Cytochrome P450; Genetic polymorphism; Interindividual variability; Racial difference; Coumarin 7-hydroxylation; Nicotine; C; -oxidation

Corrigendum to “Stimulation of neuropeptide Y-mediated calcium responses in human SMS-KAN neuroblastoma cells endogenously expressing Y2 receptors by co-expression of chimeric G proteins�? [Biochemical Pharmacology 69 (10) (2005) 1493–1499] by Frank M. Dautzenberg (pp. 809-809).

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Biochemical Pharmacology (v.70, #5)