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Biochemical Pharmacology (v.78, #8)

Editorial Advisory Board (pp. iii).

Graphical Abstracts Contents (pp. e1-e6).

Methylene blue and Alzheimer's disease by Murat Oz; Dietrich E. Lorke; George A. Petroianu (pp. 927-932).

The relationship between methylene blue (MB) and Alzheimer's disease has recently attracted increasing attention since MB has been suggested to slow down the progression of this disease.The relationship between methylene blue (MB) and Alzheimer's disease (AD) has recently attracted increasing scientific attention since it has been suggested that MB may slow down the progression of this disease. In fact, MB, in addition to its well characterized inhibitory actions on the cGMP pathway, affects numerous cellular and molecular events closely related to the progression of AD. Currently, MB has been shown to attenuate the formations of amyloid plaques and neurofibrillary tangles, and to partially repair impairments in mitochondrial function and cellular metabolism. Furthermore, various neurotransmitter systems (cholinergic, serotonergic and glutamatergic), believed to play important roles in the pathogenesis of AD and other cognitive disorders, are also influenced by MB. Recent studies suggest that the combination of diverse actions of MB on these cellular functions is likely to mediate potential beneficial effects of MB. This has lead to attempts to develop novel MB-based treatment modalities for AD. In this review article, actions of MB on neurotransmitter systems and multiple cellular and molecular targets are summarized with regard to their relevance to AD.

Keywords: Methylene blue; Alzheimer disease; Neurodegenerative diseases; Phenothiazines

The biochemical pharmacology of renin inhibitors: Implications for translational medicine in hypertension, diabetic nephropathy and heart failure: Expectations and reality by Zaid Abassi; Joseph Winaver; Giora Z. Feuerstein (pp. 933-940).

Renin, the rate-limiting enzyme in activation of the RAAS, has turned out to also be a ligand to a protein termed the renin/prorenin receptor, which binds renin and prorenin.The renin–angiotensin–aldosterone system (RAAS) plays a dominant role in the pathophysiology of hypertension, Diabetes mellitus (DM), chronic kidney disease (CKD) and chronic heart failure (CHF). Therefore, drugs that block key components of the RAAS such as ACE inhibitors (ACEi) and angiotensin receptor blockers (ARBs) have gained wide clinical use for these indications. Despite progress, the morbidity and mortality of patients treated with ACEi or ARBs remain high. Small molecules that directly inhibit renin (DRI) and are orally active have also been developed and one such drug, aliskiren, was introduced into clinical use for treatment of hypertension in 2007. Further clinical trials aimed to expand the therapeutic use of aliskiren are in progress for CKD–DM and CHF. In this review we analyze and review the translational medicine prospects of aliskiren in respect to the biochemical pharmacology of the RAAS, the marketed RAAS modulators and the new emerging science regarding the role of prorenin, renin and renin receptors in cardiovascular biology and disease. The information already gained with aliskiren, raises questions regarding the advantages of DRIs as monotherapy compared to marketed ACEis and ARBs, their potential added value in combination with other RAAS modulators and other unproven benefits in relation to prorenin and renin receptor biology. This review will also indicate basic and clinical research needs that are critical to determine whether DRIs can provide meaningful added medical benefits over contemporary medicines that regulate the RAAS, and the need to identify patients that are more likely to benefit from DRIs and any possible long term adverse effects.

Keywords: Renin; Prorenin; Direct renin inhibitors (DRI); Angiotensin I-converting enzyme (ACE); ACE inhibitors (ACEi); Angiotensin II (AngII); Angiotensin II receptors (AT1R, AT2R); Angiotensin receptor blockers (ARBs); Nitric oxide (NO); Hypertension; Diabetic nephropathy (DN); Chronic heart failure (CHF); Juxtaglomerular apparatus (JGA); Plasma renin activity (PRA); Atrial natriuretic peptide (ANP); Plasma renin activity (PRA)

A novel marine compound xyloketal B protects against oxidized LDL-induced cell injury in vitro by Wen-Liang Chen; Yan Qian; Wei-Feng Meng; Ji-Yan Pang; Yong-Cheng Lin; Yong-Yuan Guan; Sheng-Pin Chen; Jie Liu; Zhong Pei; Guan-Lei Wang (pp. 941-950).

A novel marine compound xyloketal B protects against oxidized LDL-induced endothelial cell injury through attenuating ROS generation and promoting NO release.Xyloketal B is a novel marine compound with unique chemical structure isolated from mangrove fungus Xylaria sp. (no. 2508). Pretreatment with xyloketal B (0.63–40μM) significantly improved oxLDL (150μg/ml)-induced injury in human umbilical vein endothelial cells (HUVECs) without either toxic or proliferative effects. Xyloketal B concentration-dependently attenuated oxLDL-induced ROS generation, peroxynitrite formation and decrease of Bcl-2 expression. In addition, xyloketal B significantly inhibited NADPH oxidase activity, as well as mRNA expression of gp91 phox and p47 phox. Furthermore, xyloketal B alone augmented the production of nitric oxide (NO). Collectively, these data indicate that xyloketal B protects against oxLDL-induced endothelial oxidative injury probably through inhibiting NADPH oxidase-derived ROS generation, promoting NO production and restoring Bcl-2 expression, making it a promising compound for further evaluation in the treatment of atherosclerosis.

Keywords: Abbreviations; OxLDL; oxidatively modified low-density lipoprotein; Xyl-B; xyloketal B; ROS; reactive oxygen species; NO; nitric oxide; ONOO; ⁻; peroxynitrite; EC; endothelial cell; DCFH-DA; 2′,7′-dichlorofluorescin diacetate; DAF-FM DA; 3-amino,4-aminomethyl-2′,7′-difluorescein, diacetate; HUVECs; human umbilical vein endothelial cells; eNOS; endothelial nitric-oxide synthase; L-NAME; N(G)-nitro-; l; -arginine methyl ester; 3-NT; 3-nitrotyrosine; DAPI; 4′,6-diamidino-2-phenylindole; Apo; apocyninXyloktals; Endothelial cells; Oxidized low-density lipoprotein; Reactive oxygen species; Nitric oxide; Nicotinamide adenine dinucleotide phosphate oxidase

Long term treatment with ACE inhibitor enalapril decreases body weight gain and increases life span in rats by Edson Lucas Santos; Kely de Picoli Souza; Elton Dias da Silva; Elice Carneiro Batista; Paulo J. Forcina Martins; Vânia D’Almeida; João Bosco Pesquero (pp. 951-958).

ACE inhibitor enalapril decreases body weight gain and increases life span through activation of PPARγ in the adipose tissue.Renin–angiotensin system is involved in homeostasis processes linked to renal and cardiovascular system and recently has been linked to metabolic syndrome. We analyzed the influence of long term angiotensin I converting enzyme (ACE) inhibitor enalapril treatment in normotensive adult Wistar rats fed with standard or palatable hyperlipidic diets. Our results show that long term enalapril treatment decreases absolute food intake, serum leptin concentration and body weight gain. Moreover, in adipose tissue, enalapril treatment led to decreased ACE activity, enhanced the expression of peroxisome proliferator activated receptor gamma, adiponectin, hormone-sensitive lipase, fatty acid synthase, catalase and superoxide dismutase resulting in prolonged life span. On the other hand, the ACE inhibitor was not able to improve the transport of leptin through the blood brain barrier or to alter the sensitivity of this hormone in the central nervous system. The effect of enalapril in decreasing body weight gain was also observed in older rats. In summary, these results extend our previous findings and corroborate data from the literature regarding the beneficial metabolic effects of enalapril and show for the first time that this ACE inhibitor prolongs life span in rats also fed with palatable hyperlipidic diet, an action probably correlated with adipose tissue metabolic modulation and body weight reduction.

Keywords: Abbreviations; AngII; angiotensin II; ACE; angiotensin converting enzyme; ACEi; ACE inhibitors; FAS; fatty acid synthase; GLUT4; glucose transporter-4; GPX; glutathione peroxidase; HSL; hormone-sensive lipase; icv; intracerebroventricular; PPARγ; peroxisome proliferator activated receptor gamma; RAS; renin–angiotensin system; AT; ₁; receptor type 1; Cu/Zn-SOD; copper/zinc-superoxide dismutase; Mn-SOD; manganese-superoxide dismutaseLeptin; Food intake; Obesity; RAS; Metabolic; PPARγ

PCB-induced endothelial cell dysfunction: Role of poly(ADP-ribose) polymerase by Simon G. Helyar; Bella Patel; Kevin Headington; Mary El Assal; Prabal K. Chatterjee; Pal Pacher; Jon G. Mabley (pp. 959-965).

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants implicated in the development of pro-inflammatory events critical in the pathology of atherosclerosis and cardiovascular disease. PCB exposure of endothelial cells results in increased cellular oxidative stress, activation of stress and inflammatory pathways leading to increased expression of cytokines and adhesion molecules and ultimately cell death, all of which can lead to development of atherosclerosis. To date no studies have been performed to examine the direct effects of PCB exposure on the vasculature relaxant response which if impaired may predispose individuals to hypertension, an additional risk factor for atherosclerosis. Overactivation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) following oxidative/nitrosative stress in endothelial cells and subsequent depletion of NADPH has been identified as a central mediator of cellular dysfunction. The aim therefore was to investigate whether 2,2′,4,6,6′-pentachlorobiphenyl (PCB 104) directly causes endothelial cell dysfunction via increased oxidative stress and subsequent overactivation of PARP. Exposure of ex vivo rat aortic rings to PCB 104 impaired the acetylcholine-mediated relaxant response, an effect that was dependent on both concentration and exposure time. In vitro exposure of mouse endothelial cells to PCB 104 resulted in increased cellular oxidative stress through activation of the cytochrome p450 enzyme CYP1A1 with subsequent overactivation of PARP and NADPH depletion. Pharmacological inhibition of CYP1A1 or PARP protected against the PCB 104-mediated endothelial cell dysfunction. In conclusion, the environmental contaminants, PCBs, can activate PARP directly impairing endothelial cell function that may predispose exposed individuals to development of hypertension and cardiovascular disease.

Keywords: Polychlorinated biphenyl; Oxidative stress; PARP; Cardiovascular; Atherosclerosis

Bz-423 superoxide signals B cell apoptosis via Mcl-1, Bak, and Bax by Neal B. Blatt; Anthony E. Boitano; Costas A. Lyssiotis; Anthony W. Opipari Jr.; Gary D. Glick (pp. 966-973).

Bz-423 is a pro-apoptotic 1,4-benzodiazepine with therapeutic properties in murine models of lupus demonstrating selectivity for autoreactive lymphocytes. Bz-423 modulates the F₁F₀-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. In order to understand some of the features that contribute to the increased sensitivity of lymphocytes, we report the signaling pathway engaged by Bz-423 in a Burkitt lymphoma cell line (Ramos). Following the generation of superoxide, Bz-423-induced apoptosis requires the activation of Bax and Bak to induce mitochondrial outer membrane permeabilization and cytochrome c release. Knockdown of the BH3-only proteins Bad, Bim, Bik, and Puma inhibits Bz-423 apoptosis, suggesting that these proteins serve as upstream sensors of the oxidant stress induced by Bz-423. Treatment with Bz-423 results in superoxide-dependent Mcl-1 degradation, implicating this protein as the link between Bz-423-induced superoxide and Bax and Bak activation. In contrast to fibroblasts, B cell death induced by Bz-423 is independent of c-Jun N-terminal kinase. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a specific apoptotic response that differs across cell types.

Keywords: Mcl-1; Bax; Bak; Benzodiazepine; Apoptosis; Superoxide

Distinct modes of molecular regulation of CCL3 induced calcium flux in monocytic cells by Clara Moyano Cardaba; Anja Mueller (pp. 974-982).

MCD treatment of monocytic THP-1 cells leads to an increase in the signalling properties of CCR5.The chemokine receptor CCR5 has been shown to be targeted to cholesterol- and sphingolipid-rich membrane microdomains. Here we elucidate the effects of membrane fluidity on CCR5 signalling and expression using the monocytic THP-1 cells. MCD treatment of THP-1 cells, which removes nearly all cholesterol from the plasma membrane, leads to an increase in the signalling properties of CCR5. In contrast, the prevention of cholesterol production with lovastatin and simvastatin decreases the release of intracellular calcium and also decreases receptor cell surface expression. The loss of response in lovastatin treated cells can be rescued by MCD addition, which shows that the cholesterol content in the membrane is only one factor in determining the amount of receptor response. We show that CCR5 signalling is dependent on thapsigargin-sensitive Ca²⁺ stores and on activation of ryanodine receptors as well as InsP3 receptors or store-operated channels. Cholesterol depletion with MCD changes the thapsigargin sensitivity in THP-1 cells and also alters receptor-G-protein coupling towards pertussis toxin (PTX) independent G-proteins. Cholesterol removal by MCD in THP-1 cells has far reaching consequences for receptor activation and signalling and emphasises the need for a clearer understanding of how membrane fluidity affects receptor signalling events.

Keywords: Abbreviations; CHO cells; Chinese hamster ovary cells; FCS; foetal calf serum; FITC; fluorescein isothiocyanate; HEK; human embryonic kidney; InsP3; inositol 1,4,5-trisphosphate receptor; MCD; methyl-β-cyclodextrin; PBS; phosphate buffered saline; PTX; pertussis toxin; SOCs; store-operated channelsChemokine receptor; Caveolae; Cholesterol; Microdomain; Statin

DSM-RX78, a new phosphodiesterase inhibitor, suppresses superoxide anion production in activated human neutrophils and attenuates hemorrhagic shock-induced lung injury in rats by Huang-Ping Yu; Pei-Wen Hsieh; Yi-Ju Chang; Pei-Jen Chung; Liang-Mou Kuo; Tsong-Long Hwang (pp. 983-992).

Neutrophils are activated following hemorrhagic shock and the accumulation of neutrophils in the lung is associated with lung injury. This research investigated the effects of a semisynthetic 2-benzoylaminobenzoic acid derivative, methyl 2-(2-fluorobenzamido)benzoate (DSM-RX78), on superoxide anion (O₂⁻) production in formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-activated human neutrophils, and on lung injury in Sprague–Dawley rats subjected to trauma-hemorrhage. DSM-RX78 concentration-dependently inhibited O₂⁻ production, but not elastase release, in FMLP-activated human neutrophils. DSM-RX78 displayed no superoxide-scavenging ability, and it failed to alter the subcellular NADPH oxidase activity. Significantly, DSM-RX78 increased cAMP formation and protein kinase (PK)A activity in FMLP-activated neutrophils, which occurred through the selective inhibition of cAMP-specific phosphodiesterase (PDE) activity but not an increase in adenylate cyclase function or cGMP-specific PDE activity. These results show that DSM-RX78 is a new inhibitor of cAMP-specific PDE. Moreover, DSM-RX78 reduced FMLP-induced phosphorylation of protein kinase B (Akt), but not calcium mobilization. The inhibitory effects of DSM-RX78 on O₂⁻ production and Akt phosphorylation were reversed by PKA inhibitors, suggesting that DSM-RX78 regulates O₂⁻ production of human neutrophils by promoting cAMP/PKA-dependent inhibition of Akt activation. On the other hand, administration of DSM-RX78 significantly attenuated the increase in myeloperoxidase activity and edema in the lung, as well as protein concentrations in bronchoalveolar lavage fluid in rats after trauma-hemorrhagic shock. In summary, these results strongly suggest that DSM-RX78 exerts anti-inflammatory effects, which result from the elevation of cAMP levels and PKA activity through its inhibition of cAMP-specific PDE. Also, our findings show that DSM-RX78 attenuates hemorrhagic shock-induced lung injury in rats.

Keywords: Abbreviations; AC; adenylyl cyclase; Akt; protein kinase B; ARDS; acute respiratory distress syndrome; cAMP; cyclic adenosine 3′,5′-monophosphate; CB; cytochalasin B; COPD; chronic obstructive pulmonary disease; FMLP; formyl-; l; -methionyl-; l; -leucyl-; l; -phenylalanine; GPCR; G protein-coupled receptor; H89; N; -(2-((; p; -bromocinnamyl)amino)ethyl)-5-isoquinolinesulfonamide; IBMX; 3-isobutyl-1-methylxanthine; KT5720; 9S,10S,12R-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo(1,2,3-fg:3′,2′,1′-kl)pyrrolo(3,4-i)(1,6)benzodiazocine-10-carboxylic acid hexyl ester; MPO; myeloperoxidase; O; ₂; ⁻; superoxide anion; PDE; phosphodiesterase; PKA; protein kinase A; PKC; protein kinase C; LDH; lactate dehydrogenase; PMA; phorbol myristate acetate; Ro318220; 3-(1-(3-(amidinothio)propyl-1H-indol-3-yl))-3-(1-methyl-1H-indol-3-yl)maleimide; SOD; superoxide dismutase; SPA; scintillation proximity assay; WST-1; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium saltcAMP; Hemorrhagic shock; Human neutrophils; Methyl 2-(2-fluorobenzamido)benzoate; Phosphodiesterase; Superoxide anion

Pharmacology of AMD3465: A small molecule antagonist of the chemokine receptor CXCR4 by Veronique Bodart; Virginia Anastassov; Marilyn C. Darkes; Stefan R. Idzan; Jean Labrecque; Gloria Lau; Renee M. Mosi; Kathleen S. Neff; Kim L. Nelson; Melanie C. Ruzek; Ketan Patel; Zefferino Santucci; Robert Scarborough; Rebecca S.Y. Wong; Gary J. Bridger; Ron T. MacFarland; Simon P. Fricker (pp. 993-1000).

AMD3465 is a selective antagonist of the chemokine receptor CXCR4 which exhibits biphasic pharmacokinetics and causes rapid leukocytosis, a surrogate for hematopoietic stem cell mobilization, when administered subcutaneously.CXCR4 is widely expressed in multiple cell types, and is involved in neonatal development, hematopoiesis, and lymphocyte trafficking and homing. Disruption of the CXCL12/CXCR4 interaction has been implicated in stem cell mobilization. Additionally CXCR4 is a co-receptor for HIV. Selective small molecule antagonists of CXCR4 therefore have therapeutic potential. AMD3465 is an N-pyridinylmethylene monocyclam CXCR4 antagonist which can block infection of T-tropic, CXCR4-using HIV. Using the CCRF-CEM T-cell line which expresses CXCR4 we have demonstrated that AMD3465 is an antagonist of SDF-1 ligand binding ( K_i of 41.7±1.2nM), and inhibits SDF-1 mediated signaling as shown by inhibition of GTP binding, calcium flux, and inhibition of chemotaxis. AMD3465 is selective for CXCR4 and does not inhibit chemokine-stimulated calcium flux in cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7, nor does it inhibit binding of LTB₄ to its receptor, BLT1. The pharmacokinetics of AMD3465 was investigated in mice and dogs. Absorption was rapid following subcutaneous administration. AMD3465 was cleared from dog plasma in a biphasic manner with a terminal half-life of 1.56–4.63h. Comparison of exposure to the intravenous and subcutaneous doses indicated 100% bioavailability following subcutaneous administration. AMD3465 caused leukocytosis when administered subcutaneously in mice and dogs, with peak mobilization occurring between 0.5 and 1.5h following subcutaneous dosing in mice and with maximum peak plasma concentration of compound preceding peak mobilization in dogs, indicating that AMD3465 has the potential to mobilize hematopoietic stem cells. These data demonstrate the therapeutic potential for the CXCR4 antagonist AMD3465.

Keywords: AMD3465; Chemokine receptor; CXCR4; Leukocytosis; Pharmacokinetics

Ovariectomy and estrogen treatment modulate iron metabolism in rat adipose tissue by Giuseppina Mattace Raso; Carlo Irace; Emanuela Esposito; Carmen Maffettone; Anna Iacono; Antonio Di Pascale; Rita Santamaria; Alfredo Colonna; Rosaria Meli (pp. 1001-1007).

Iron is essential for many biological processes and its deficiency or excess is involved in pathological conditions. At cellular level, the maintenance of iron homeostasis is largely accomplished by the transferrin receptor (TfR-1) and by ferritin, whose expression is mainly regulated post-transcriptionally by iron regulatory proteins (IRPs).This study examines the hypothesis that modification of serum estrogen levels by ovariectomy and 17β-estradiol (E₂) treatment in rats modulate serum iron-status parameters and iron metabolism in adipose tissue. In particular, we evaluated the RNA binding of IRP1 by electrophoretic mobility-shift assay and IRP1, ferritin, and TfR-1 expression in adipose tissue by Western blot analysis.Ovariectomy, besides a lowered serum iron and transferrin iron binding capacity, remarkably decreased the binding activity of IRP1 in peritoneal and subcutaneous adipose tissues, and these effects were reversed by E₂ treatment. Moreover, ovariectomy determined a decrease of IRP1 expression, which was significant in subcutaneous adipose tissue. Consistent with IRP1 regulation, an increase of ferritin and a decrease of TfR-1 expression were observed in peritoneal adipose tissue from ovariectomized animals, while the treatment with E₂ reconstituted TfR-1 level. A similar expression profile of TfR-1 was observed in subcutaneous adipose tissue, where ferritin level did not change in ovariectomized animals, and was increased after E₂ treatment.Our results indicate that estrogen level changes can regulate the binding activity of the IRP1, and consequently ferritin and TfR-1 expression in adipose tissue, suggesting a relationship among serum and tissue iron parameters, estrogen status and adiposity.

Keywords: Estrogen; Ovariectomy; Adipose tissue; Iron metabolism; Iron regulatory proteins

Fatty acid derivatised analogues of glucose-dependent insulinotropic polypeptide with improved antihyperglycaemic and insulinotropic properties by Barry D. Kerr; Nigel Irwin; Finbarr P.M. O’Harte; Clifford J. Bailey; Peter R. Flatt; Victor A. Gault (pp. 1008-1016).

C-terminal acylation of Lys³⁷ with myristic (MYR; tetradecanoic acid), palmitic (PAL; hexadecanoic acid) and stearic (octadecanoic acid) fatty acids with or without N-terminal acetylation was employed to develop long-acting analogues of the glucoregulatory hormone, glucose-dependent insulinotropic polypeptide (GIP). All GIP analogues exhibited resistance to dipeptidylpeptidase-IV (DPP-IV) and significantly improved in vitro cAMP production and insulin secretion. Administration of GIP analogues to ob/ob mice significantly lowered plasma glucose—GIP(Lys³⁷MYR), N-AcGIP(Lys³⁷MYR) and GIP(Lys³⁷PAL) increased plasma insulin concentrations. GIP(Lys³⁷MYR) and N-AcGIP(Lys³⁷MYR) elicited protracted glucose-lowering effects when administered 24h prior to an intraperitoneal glucose load. Daily administration of GIP(Lys³⁷MYR) and N-AcGIP(Lys³⁷MYR) to ob/ob mice for 24 days decreased glucose and significantly improved plasma insulin, glucose tolerance and beta-cell glucose responsiveness. Insulin sensitivity, pancreatic insulin content and triglyceride levels were not changed. These data demonstrate that C-terminal acylation particularly with myristic acid provides a class of stable, longer-acting forms of GIP for further evaluation in diabetes therapy.

Keywords: Dipeptidylpeptidase-IV (DPP-IV); Glucose-dependent insulinotropic polypeptide (GIP); GIP agonist; Glucose homeostasis; Insulin secretion

Phosphorylation of human enhancer of filamentation (HEF1) on serine 369 induces its proteasomal degradation by Virginie Hivert; Josiane Pierre; Joël Raingeaud (pp. 1017-1025).

Human enhancer of filamentation 1 (HEF1) is a multi-domain docking protein of the p130 Cas family. HEF1 is present at focal adhesions and is involved in integrin signalling mediating cytoskeleton reorganization associated with cell migration, adhesion or apoptosis. HEF1 functions are regulated in part by phosphorylation on tyrosine residues. HEF1 is also phosphorylated on serines/threonines leading to two isoforms refered to as p105 and p115. In most cases, the serine/threonine kinase(s) responsible for HEF1 phosphorylation have not been identified. In the present study, we have investigated HEF1 ser/thr phosphorylation. In the HCT-116 cell line transiently overexpressing Flag-HEF1 we showed that Hesperadin, a synthetic indolinone displaying antiproliferative effect and described as an inhibitor of various kinases including Aurora-B, prevented HEF1 phosphorylation induced by the ser/thr phosphatase PP2A inhibitor: okadaic acid (OA). In addition we showed that conversion of endogenous HEF1 p105 to p115 in HaCaT cells was prevented upon treatment with Hesperadin, resulting in accumulation of p105HEF1. We also identified serine 369 as the target site of phosphorylation by this Hesperadin-inhibited kinase in HCT-116. Finally, we provide evidence that phosphorylation on serine 369 but not phosphorylation on serine 296, triggers HEF1 degradation by the proteasomal machinery. These data suggest that conversion of p105 to p115 results from a ser-369-dependent phosphorylation mediated by an Hesperadin-sensitive kinase and regulates the half-life of HEF1.

Keywords: Abbreviations; HEF1; human enhancer of filamentation 1; FAK; focal adhesion kinase; CrkL; Crk-like; PP; protein phosphatase; Cas; Crk associated substrate; SH; Src homology; APC; anaphase promoting complexHuman enhancer of filamentation 1; Protein phosphatase 2A; Hesperadin; Protein stability

Spinal ERK activation via NO–cGMP pathway contributes to nociceptive behavior induced by morphine-3-glucuronide by Takaaki Komatsu; Shinobu Sakurada; Kazuhiro Kohno; Hideo Shiohira; Sou Katsuyama; Chikai Sakurada; Minoru Tsuzuki; Tsukasa Sakurada (pp. 1026-1034).

Intrathecal (i.t.) injection of morphine-3-glucuronide (M3G), a major metabolite of morphine without analgesic actions, produces a severe hindlimb scratching followed by biting and licking in mice. The pain-related behavior evoked by M3G was inhibited dose-dependently by i.t. co-administration of tachykinin NK₁ receptor antagonists, sendide, [D-Phe⁷, D-His⁹] substance P(6-11), CP-99994 or RP-67580 and i.t. pretreatment with antiserum against substance P. The competitive NMDA receptor antagonists, D-APV and CPP, the NMDA ion-channel blocker, MK-801 or the competitive antagonist of the polyamine recognition site of NMDA receptor ion-channel complex, ifenprodil, produced inhibitory effects on i.t. M3G-evoked nociceptive response. The NO–cGMP–PKG pathway, which involves the extracellular signal-regulated kinase (ERK), has been implicated as mediators of plasticity in several pain models. Here, we investigated whether M3G could influence the ERK activation in the NO–cGMP–PKG pathway. The i.t. injection of M3G evoked a definite activation of ERK in the lumbar dorsal spinal cord, which was prevented dose-dependently by U0126, a MAP kinase-ERK inhibitor. The selective nNOS inhibitor N^ω-propyl-l-arginine, the selective iNOS inhibitor W1400, the soluble guanylate cyclase inhibitor ODQ and the PKG inhibitor KT-5823 inhibited dose-dependently the nociceptive response to i.t. M3G. In western blotting analysis, inhibiting M3G-induced nociceptive response using these inhibitors resulted in a significant blockade of ERK activation induced by M3G in the spinal cord. Taken together, these results suggest that activation of the spinal ERK signaling in the NO–cGMP–PKG pathway contributes to i.t. M3G-evoked nociceptive response.

Keywords: Morphine-3-glucuronide (M3G); NK; ₁; receptor; NMDA receptor; Nitric oxide (NO)–cGMP–PKG pathway; Extracellular signal-regulated kinase (ERK); Dorsal spinal cord; Mice

Dimebolin is a 5-HT₆ antagonist with acute cognition enhancing activities by Hervé Schaffhauser; Joanne R. Mathiasen; Amy DiCamillo; Mark J. Huffman; Lily D. Lu; Beth A. McKenna; Jie Qian; Michael J. Marino (pp. 1035-1042).

Dimebolin (Dimebon™), is a non-selective antihistamine approved in Russia for the treatment of allergy. Recently, this drug has been shown to be neuroprotective in cellular models of Alzheimer's disease and Huntington's disease, and to preserve cognitive function when chronically administered to AF64A lesioned rats. Interests in identifying the molecular targets of dimebolin have intensified with reports of efficacy in clinical trials with Alzheimer's patients. Dimebolin has been found to interact with a number of molecular targets including acetylcholinesterases, N-methyl-d-aspartate receptors, and voltage-gated calcium channels, with potencies in the range of 5–50μM. In the present study, the action of dimebolin at the serotonin 5-HT₆ receptor was investigated. Dimebolin binds with moderate affinity to both the human and rat recombinant 5-HT₆ receptor ( K_i=26.0±2.5nM and 119.0±14.0nM respectively) as well as the native rat 5-HT₆ receptor, and acts as an antagonist in functional cAMP assays. Furthermore, dimebolin occupies the 5-HT₆ receptor in vivo as assessed by ex vivo autoradiography, with a dose–occupancy relationship similar to that of the selective 5-HT₆ antagonist SB-399885. Finally, both SB-399885 and dimebolin produce an acute enhancement of short-term social recognition memory, although dimebolin is approximately 10-fold less potent than SB-399885. Taken together, these studies demonstrate that dimebolin antagonizes the 5-HT₆ receptor with higher affinity than other targets characterized to date, and suggest that this activity may play a role in the acute cognition enhancing effects of this compound in preclinical models and in the clinic.

Keywords: Abbreviations; Dimebolin; 2,8-Dimethyl-5-[2-(6-methylpyridin-3-yl)ethyl]-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole dihydrochloride; SB-399885; (N-(3,5-Dichloro-2-methoxyphenyl)-4-methoxy-3-piperazin-1-yl-benzenesulfonamide hydrochloride; SB-258528; 4-Iodo-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]benzene-sulfonamide; NMDA; N-methyl-; d; -aspartate receptors; 5-HT; serotonin; AD; Alzheimer disease; ANOVA; analysis of varianceDimebolin; Dimebon; 5-HT; ₆; Cognition; Alzheimer's disease

Species differences in pharmacokinetic and pharmacodynamic properties of nebicapone by Maria João Bonifácio; Ana I. Loureiro; Leonel Torrão; Carlos Fernandes-Lopes; Lyndon Wright; Maria João Pinho; Patrício Soares-da-Silva (pp. 1043-1051).

The present study was designed to characterize pharmacodynamic and pharmacokinetic properties of nebicapone in rats and mice. Upon oral administration of nebicapone the extent of mouse liver catechol-O-methyltransferase (COMT) inhibition is half that in the rat. Nebicapone was rapidly absorbed reaching plasma C_max within 30min and being completely eliminated by 8h. Nebicapone was metabolized mainly by glucuronidation and methylation in both species, but rat had an extra major metabolite, resulting from sulphation. Administration of nebicapone by the intraperitoneal route significantly increased compound AUC in the rat while in the mouse a significant increase in AUC of metabolites was observed. These results show that nebicapone exhibited marked species differences in bioavailability and metabolic profile. Evaluation of COMT activity in rat and mice liver homogenates revealed that both had similar methylation efficiencies ( K_cat values, respectively 7.3 and 6.4min⁻¹), but rat had twice active enzyme units as the mouse (molar equivalency respectively 150 and 83). Furthermore, nebicapone inhibited rat liver COMT with a lower K_i than mouse liver COMT (respectively 0.2nM vs. 1.2nM). In conclusion, the results from the present study show that mice and rats respond differently to COMT inhibition by nebicapone. The more pronounced inhibitory effects of nebicapone in the rat may be related to an enhanced oral availability and less pronounced metabolism of nebicapone in this specie, but also concerned with the predominant expression of S-COMT over MB-COMT, the latter of which is less sensitive to inhibition by nebicapone than the former.

Keywords: COMT; Nebicapone; Species differences; Rat; Mouse; Kinetics; Drug metabolism

Regional differences in capillary density, perfusion rate, and P-glycoprotein activity: A quantitative analysis of regional drug exposure in the brain by Rong Zhao; Gary M. Pollack (pp. 1052-1059).

The rate of regional perfusion flow (diazepam as marker), as well as P-gp-mediated colchicine efflux activity, was directly proportional to local capillary density (inulin as marker) in murine brain.The in situ brain perfusion technique was used to assess the impact of local capillary density, blood flow rate and P-gp-mediated efflux activity on regional drug exposure for the P-gp substrates colchicine, quinidine, verapamil, and loperamide, the perfusion flow rate marker diazepam, and the vascular volume marker inulin, in mdr1a(+/+) and mdr1a(−/−) mice. Regional perfusion flow rate varied 7.5-fold, and capillary density (based on vascular volume) varied 3.7-fold, across the 13 brain regions examined. The rate of regional flow, as well as P-gp-mediated colchicine efflux activity, was directly proportional to local capillary density. A decrease in perfusion rate attenuated verapamil brain uptake and had significant effect on P-gp-mediated efflux activity for this substrate in brain regions with lower capillary density. Regional brain uptake and calculated log D at pH 7.4 (clog D_7.4) were well-related in P-gp-deficient mice, indicating that in the absence of P-gp-mediated efflux, physicochemical properties of the compound (i.e., lipophilicity) serve as the primary determinant of regional brain uptake. Loperamide regional brain uptake and P-gp effect during a 60-s brain perfusion or at 30min after subcutaneous administration were significantly correlated with local capillary density. The highest P-gp-mediated efflux activity was consistently observed in cerebral cortex and midbrain regions for loperamide following short-term brain perfusion and at all time points following subcutaneous administration. These results in intact animal emphasize that the regionality of substrate exposure in brain as measured by the in situ brain perfusion technique is actually biologically relevant.

Keywords: Abbreviations; BBB; blood–brain barrier; CNS; central nervous system; MTT; mean transit time; P-gp; P-glycoproteinBrain capillary density; Perfusion flow rate; P-glycoprotein; Regional drug distribution

Effect of progesterone and its synthetic analogues on the activity of mitochondrial permeability transition pore in isolated rat liver mitochondria by Nadezhda I. Fedotcheva; Vera V. Teplova; Tatiana A. Fedotcheva; Vladimir M. Rzheznikov; Nikolai L. Shimanovskii (pp. 1060-1068).

The influence of progesterone and its synthetic analogues on the induction of the Ca²⁺-dependent mitochondrial permeability transition pore (MPTP) has been studied. The novel synthetic analogue of progesterone 17a-acetoxy-3b-butanoyloxy-6-methyl-pregna-4,6-diene-20-on (buterol) was compared with progesterone and medroxyprogesterone acetate (MPA). It was found that progesterone and buterol have opposite effects on the induction of MPTP opening by calcium ions. By contrast to progesterone, which decreased the calcium ion concentration necessary for pore opening, and MPA, which also, although at a lesser extent, activated the pore induction, buterol at a concentration of 20–100μM blocked the pore opening and increased the calcium retention capacity of mitochondria more than twofold. The action of buterol is specific to the pore since it did not affect the respiration, whereas progesterone completely inhibited NAD-dependent respiration. MPA acted similar to progesterone but less effectively. The inhibitory effect of buterol was eliminated in the presence of carboxyatractyloside, which selectively binds the thiol groups of adenylate translocase and prevents the adenine nucleotide binding. These data indicate that buterol interacts with thiol groups, which explains its inhibitory effect not only on the mitochondrial pore but also on the transport system of xenobiotics in tumor cells in which buterol reduces the multidrug resistance.

Keywords: Abbreviations; ANT; adenine nucleotide translocase; BuOOH; t; -butylhydroperoxide; Buterol; 17a-acetoxy-3b-butanoyloxy-6-methyl-pregna-4,6-diene-20-on; CAT; carboxyatractyloside; MPA; medroxyprogesterone acetate; MPTP; mitochondrial permeability transition pore; NEM; N; -ethylmaleimide; Δ; Ψ; mitochondrial membrane potentialProgesterone; Medroxyprogesterone acetate; Buterol; Mitochondrial permeability transition pore; Thiol groups

Epigallocatechin-3-gallate is an inhibitor of Na⁺,K⁺-ATPase by favoring the E₁ conformation by Hideo Ochiai; Kazuo Takeda; Shiori Soeda; Yoshikazu Tahara; Hitoshi Takenaka; Kazuhiro Abe; Yutaro Hayashi; Shunsuke Noguchi; Masumi Inoue; Silvia Schwarz; Wolfgang Schwarz; Masaru Kawamura (pp. 1069-1074).

Four catechins, epigallocatechin-3-gallate, epigallocatechin, epicatechin-3-gallate, and epicatechin, inhibited activity of the Na⁺,K⁺-ATPase. The two galloyl-type catechins were more potent inhibitors, with IC₅₀ values of about 1μM, than were the other two catechins. Inhibition by epigallocatechin-3-gallate was noncompetitive with respect to ATP. Epigallocatechin-3-gallate reduced the affinity of vanadate, shifted the equilibrium of E₁P and E₂P toward E₁P, and reduced the rate of the E₁P to E₂P transition. Epigallocatechin-3-gallate potently inhibited membrane-embedded P-type ATPases (gastric H⁺,K⁺-ATPase and sarcoplasmic reticulum Ca²⁺-ATPase) as well as the Na⁺,K⁺-ATPase, whereas soluble ATPases (bacterial F₁-ATPase and myosin ATPase) were weakly inhibited. Solubilization of the Na⁺,K⁺-ATPase with a nonionic detergent reduced sensitivity to epigallocatechin-3-gallate with an elevation of IC₅₀ to 10μM. These results suggest that epigallocatechin-3-gallate exerts its inhibitory effect through interaction with plasma membrane phospholipid.

Keywords: Epigallocatechin-3-gallate; Na; ⁺; ,K; ⁺; -ATPase; Inhibition; P-type ATPase; Phospholipid

Clock gene mutation modulates the cellular sensitivity to genotoxic stress through altering the expression of N-methylpurine DNA glycosylase gene by Jahye Kim; Naoya Matsunaga; Satoru Koyanagi; Shigehiro Ohdo (pp. 1075-1082).

CLOCK protein acts as a positive regulator for transcription of N-methylpurine DNA glycosylase gene.Although Clock gene product, a component of the circadian pacemaker, has been suggested to participate in the regulation of cellular sensitivity to genotoxic stress, the underlying mechanism remains to be fully understood. In this study, we showed that Clock gene mutation modulates the sensitivity of hepatocytes to alkylating agent-induced genotoxic stress through altering the expression of N-methylpurine DNA glycosylase (MPG), the first enzyme in the base excision repair pathway. Neither wild-type nor Clock mutant ( Clock/ Clock) mice showed a significant 24-h variation in the hepatic expression of MPG. However, the mRNA and protein levels of MPG in the liver of Clock/ Clock mice were significantly lower than those in wild-type liver. The cytotoxic effect of methyl methanesulfonate (MMS), a methylating agent, on primary cultured hepatocytes prepared from Clock/ Clock mice was more potent than on wild-type hepatocytes, while overexpression of MPG in Clock/ Clock hepatocytes restored their MMS sensitivity to the wild-type level. These findings suggest that the product of the Clock gene controls the sensitivity of cells to genotoxic stress through regulating the expression of the MPG gene. Our present findings would provide a molecular link between the circadian clock and DNA repair pathway.

Keywords: Abbreviations; ALT; alanine transaminase; AST; aspartate transaminase; APEX1; abasic endonuclease 1; BER; base excision repair; LDH; lactate dehydrogenase; LIG1; DNA ligase 1; MGMT; O; ⁶; -methylguanine-DNA methyltransferase; MMS; methyl methansulfonate; MPG; N-methylpurine-DNA glycosylase; MSH2; mutS homolog 2; MSH6; mutS homolog 2; MLH1; mutL homolog 1; PMS2; postmeiotic segregation increased 2; POLB; polymerase beta; UDG; uracil-DNA glycosylase; 8oxoDG; 8-oxoguanine-DNA glycosylaseClock gene; DNA repair; N-methylpurine DNA glycosylase; Alkylating agent; Methyl methanesulfonate

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Biochemical Pharmacology (v.78, #8)

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Biochemical Pharmacology (v.78, #8)