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Biochemical Pharmacology (v.75, #7)

Editorial Advisory Board (pp. iii).

Expression, regulation and function of trail in atherosclerosis by Mary M. Kavurma; Martin R. Bennett (pp. 1441-1450).

Atherosclerosis is a condition where vascular smooth muscle cells (VSMCs), inflammatory cells, lipids, cholesterol and cellular waste accumulate in the inner lining of an artery, producing a fibro-fatty plaque and resulting in the thickening of the arterial wall. The tumor necrosis factor (TNF) family of cytokines plays a major role in the progression of atherosclerosis. Recently, TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, has been implicated in the development of atherosclerosis since it has been detected in normal and diseased atherosclerotic tissue. Not only is TRAIL involved in apoptosis and immune regulation, recent studies have provided a new function of TRAIL on vascular cells, such that TRAIL can promote endothelial cell (EC) and VSMCs migration and proliferation. In addition, TRAIL is implicated in regulating vascular tone. This review discusses our current understanding of TRAIL expression, regulation and function, and summarises the recent data implicating a role for TRAIL in atherosclerosis.

Mechanisms of cell death induced by 2-chloroadenosine in leukemic B-cells by Laurent Bastin-Coyette; Caroline Smal; Sabine Cardoen; Pascale Saussoy; Eric Van Den Neste; Françoise Bontemps (pp. 1451-1460).

2-Chloroadenosine (2-CAdo) is an adenosine deaminase-resistant analogue of adenosine, widely used as an adenosine receptor agonist. This compound has been shown to induce apoptosis in several cell types either via activation of adenosine receptors or via intracellular metabolism. However, the molecular mechanisms of 2-CAdo-induced apoptosis are unclear. Here, we analyzed the effects of 2-CAdo in the leukemia cell line EHEB. 2-CAdo was found to induce apoptosis in EHEB cells, as shown by caspase-3 activation, DNA fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage and phosphatidylserine exposure. Cytotoxicity of 2-CAdo was completely suppressed by 5-iodotubercidin, an adenosine kinase inhibitor, indicating that apoptosis induced by 2-CAdo was the result of its intracellular metabolism. Accordingly, we found that 2-CAdo was efficiently converted into 2-chloroATP. In parallel, a decrease of intracellular ATP concentration as well as a general inhibition of macromolecular synthesis, involving DNA, RNA and protein synthesis, was observed. Moreover, 2-CAdo induced cytochrome c release into the cytosol, indicating activation of the intrinsic pathway of apoptosis. This was found associated with a decline in Mcl-1 protein level and p53-independent. Inhibition of AMP deaminase by coformycin markedly prevented ATP depletion, and also significantly reduced 2-CAdo cytotoxicity and caspase-3 activation. In conclusion, our data show that intracellular metabolism of 2-CAdo can lead to activation of the intrinsic pathway of apoptosis and that ATP depletion, in addition to the accumulation of the triphosphate analogue, contributes to 2-CAdo-induced apoptosis.

Keywords: Abbreviations; 2-CAdo; 2-chloroadenosine; CdA; 2-chloro-2′-deoxyadenosine; 2-CATP; 2-chloroATP; CLL; chronic lymphocytic leukaemia; HPLC; high-performance liquid chromatography; ITu; 5-iodotubercidin; NBTI; nitrobenzylthioinosine; PARP; poly(ADP-ribose) polymerase; PI; propidium iodide2-Chloroadenosine; Mitochondrial pathway; ATP; Mcl-1; B-CLL

Dimeric procyanidin B2 inhibits constitutively active NF-κB in Hodgkin's lymphoma cells independently of the presence of IκB mutations by Gerardo G. Mackenzie; Ana M. Adamo; Noah P. Decker; Patricia I. Oteiza (pp. 1461-1471).

Due to long-term toxicity of current Hodgkin's lymphoma (HL) treatment, the present challenge is to find new therapies that specifically target deregulated signaling cascades, including NF-κB, which are involved in Hodgkin (H) and Reed-Sternberg (RS) cell proliferation and resistance to apoptosis. We previously presented evidence that dimeric procyanidin B2 (B2) can interact with NF-κB proteins inhibiting the binding of NF-κB to DNA. Herein, we investigated if B2, acting at a late event in NF-κB signaling cascade, could be effective in inhibiting NF-κB in H–RS cells with different mechanisms of constitutive NF-κB activation. B2 caused a concentration-dependent inhibition of NF-κB-DNA binding to a similar extent (41–48% inhibition at 25μM B2) in all the tested H–RS cell lines (L-428, KM-H2, L-540, L-1236 and HDML-2). This was associated with the inhibition of NF-κB-driven gene expression, including cytokines (IL-6, TNFα and RANTES) and anti-apoptotic proteins (Bcl-xL, Bcl-2, XIAP and cFLIP). The finding of similar amounts of RelA and p50 proteins in the nucleus, but decreased NF-κB-DNA binding, even in those H–RS cells characterized by mutations in the inhibitory IκB proteins, supports that B2 acts by preventing the binding of NF-κB to DNA. B2 did not inhibit AP-1 and STAT3 constitutive activation in H–RS cells, indicating that the moderate effects of B2 on cell viability are due to the complex signaling aberrations in HL. Thus, several signaling pathways should be targeted when designing therapeutics for HL. In this regard, the capacity of B2 to inhibit NF-κB could be valuable in a multi-drug approach.

Keywords: Abbreviations; DCDHF; 5(or 6)-carboxy-2′7′-dichlorodihydrofluorescein diacetate; DCF; 2′7′-dichlorofluorescein; DTT; dithiothreitol; EBV; Epstein-Barr virus; EDTA; ethylenediamine tetraacetate; EMSA; electrophoretic mobility shift assay; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HL; Hodgkin's lymphoma; H–RS cells; Hodgkin and Reed-Sternberg cells; IKK; IκB kinase; IL-6; interleukin-6; LMP-1; latent membrane protein-1; NF-κB; nuclear factor-κB; PBS; phosphate-buffered saline; RANTES; Regulated on Activation Normal T cell Expressed and Secreted; TNFα; tumor necrosis factor alphaHodgkin's lymphoma; NF-κB; Procyanidin B2; Flavanols; H–RS cells

Acetylation of prostaglandin H₂ synthases by aspirin is inhibited by redox cycling of the peroxidase by Manju Bala; Cindy N. Chin; Asha T. Logan; Taneem Amin; Lawrence J. Marnett; Olivier Boutaud; John A. Oates (pp. 1472-1481).

Aspirin exerts its unique pharmacological effects by irreversibly acetylating a serine residue in the cyclooxygenase site of prostaglandin-H₂-synthases (PGHSs). Despite the irreversibility of the inhibition, the potency of aspirin varies remarkably between cell types, suggesting that molecular determinants could contribute to cellular selectivity. Using purified enzymes, we found no evidence that aspirin is selective for either of the two PGHS isoforms, and we showed that hydroperoxide substrates of the PGHS peroxidase inhibited the rate of acetylation of PGHS-1 by 68%. Using PGHS-1 reconstituted with cobalt protoporphyrin, a heme devoid of peroxidase activity, we demonstrated that reversal by hydroperoxides of the aspirin-mediated acetylation depends upon the catalytic activity of the PGHS peroxidase. We demonstrated that inhibition of PGHS-2 by aspirin in cells in culture is reversed by 12-hydroperoxyeicosatetraenoic acid dose-dependently (ED₅₀=0.58±0.15μM) and that in cells with high levels of hydroperoxy-fatty acids (RAW264.7) the efficacy of aspirin is markedly decreased as compared to cells with low levels of hydroperoxides (A549; IC₅₀s=256±22μM and 11.0±0.9μM, respectively). Together, these findings indicate that acetylation of the PGHSs by aspirin is regulated by the catalytic activity of the peroxidase, which yields a higher oxidative state of the enzyme.

Keywords: Abbreviations; PGHS; prostaglandin H; ₂; synthase; PG; prostaglandin; 12-HPETE; 12-hydroperoxyeicosatetraenoic acid; 12-HETE; 12-hydroxyeicosatetraenoic acid; PPHP; 5-phenyl-4-pentenyl hydroperoxide; PPA; 5-phenyl-4-pentenyl alcohol; DMEM; Dulbecco's modified Eagle's medium; FBS; foetal bovine serum; Tris; tris(hydroxymethyl) aminomethane; FePPIX; iron protoporphyrin IX; AA; arachidonic acid; LPS; lipopolysaccahrideProstaglandin H synthase; Cyclooxygenase; Aspirin; Hydroperoxide; Cellular specificity

Increased glycogen stores due to γ-AMPK overexpression protects against ischemia and reperfusion damage by Michal Ofir; Michael Arad; Eyal Porat; Dov Freimark; Yelena Chepurko; Bernardo A. Vidne; Christine E. Seidman; Jonathan G. Seidman; Bruce E. Kemp; Edith Hochhauser (pp. 1482-1491).

During ischemia, endogenous glycogen becomes the principal substrate for energy through glycolysis. Cardiac-specific manipulation of AMP-activated protein kinase (AMPK) by over-expression of its regulatory γ-subunit induces glycogen storage. The aim of this study was to examine whether heart glycogen in transgenic mice overexpressing PRKAG2 may protect from ischemia and reperfusion injury. Isolated hearts were mounted on Langendorff apparatus and subjected to 30min ‘no-flow’ or ‘low-flow’ ischemia and 60min reperfusion. Hemodynamic measurements, tetrazolium staining, glycogen and lactate were used to monitor ischemia reperfusion damage. After low-flow ischemia, left ventricular pressure, coronary flow (CF) and the area of viable myocardium were 20–30% higher in PRKAG2 mice compared to controls. The basal levels of glycogen in PRKAG2 were 9.2μg/g, markedly higher than in controls, but after low-flow ischemia they declined concomitantly with increased lactate washout in the coronary effluent. During no-flow ischemia there was neither protection nor consumption of glycogen in PRKAG2 hearts. Cardioprotection was also eliminated when PRKAG2 hearts were depleted of glycogen prior to low-flow ischemia. AMPK α Thr¹⁷² phosphorylation did not differ between PRKAG2 hearts and controls either during low-flow ischemia or reperfusion. We conclude that PRKAG2 hearts resist low-flow ischemia injury better than controls. Improved recovery was associated with increased consumption of glycogen, and was unrelated to AMPK activation. These findings demonstrate the potential of heart protection from ischemia and reperfusion injury through metabolic manipulation increasing the level and utilization of myocardial glycogen.

Keywords: Ischemia reperfusion; Low-flow ischemia; Glycogen; AMP-activated protein kinase; Myocardium; Lactate

The high-affinity immunoglobulin E receptor (FcɛRI) regulates mitochondrial calcium uptake and a dihydropyridine receptor-mediated calcium influx in mast cells: Role of the FcɛRIβ chain immunoreceptor tyrosine-based activation motif by Yoshihiro Suzuki; Tetsuro Yoshimaru; Toshio Inoue; Satoshi Nunomura; Chisei Ra (pp. 1492-1503).

A growing body of evidence suggests that mitochondria take up calcium upon receptor (agonist) stimulation and that this contributes to the dynamics of spatiotemporal calcium signaling. We have previously shown that engagement of the high-affinity receptor for immunoglobulin E (FcɛRI) stimulates mitochondrial calcium ([Ca²⁺]_m) uptake in mast cells. The present study was undertaken to investigate the mechanisms and biological significance of FcɛRI regulation of [Ca²⁺]_m. Antigen stimulated [Ca²⁺]_m uptake in a dose-dependent manner with a minimal effective dose of 0.03–3ng/ml. This [Ca²⁺]_m uptake took place immediately, reaching its peak within minutes and was inhibited by the src family kinase inhibitor PP1 and phosphatidylinositol-3-kinase inhibitor wortmannin. Analyses using mast cells expressing the wild-type or the mutated type of the FcɛRIβ immunoreceptor tyrosine-based activation motif (ITAM) in which all tyrosine residues were replaced by phenylalanine revealed that the FcɛRIβ ITAM is essential for a sustained [Ca²⁺]_m uptake. The FcɛRIβ ITAM was essential for overall calcium response upon weak FcɛRI stimulation (at low antigen concentration), while upon strong stimulation (at high antigen concentration) it appeared necessary selectively to an immediate calcium response that was sensitive to the dihydropyridine receptor (DHPR) antagonist nifedipine and wortmannin but not to the store-operated calcium entry (SOCE) antagonists such as 2-aminoethoxyphenyl borate and SK&F96365. These data demonstrate that the FcɛRIβ regulates [Ca²⁺]_m uptake in mast cells via the ITAM and suggest that this plays a key role in regulating calcium influx especially that induced via a DHPR-mediated calcium channel.

Keywords: Mast cells; FcɛRI; Calcium; Mitochondria; Dihydropyridine receptor

Sp1 is an essential transcription factor for LPS-induced tissue factor expression in THP-1 monocytic cells, and nobiletin represses the expression through inhibition of NF-κB, AP-1, and Sp1 activation by Yuki Hirata; Yutaka Masuda; Hideki Kakutani; Toshiyuki Higuchi; Kimihiko Takada; Akira Ito; Yoshio Nakagawa; Hidemi Ishii (pp. 1504-1514).

Nobiletin is a citrus polymethoxylated flavonoid extracted from Citrus depressa, and has several reported biological effects. In this study, we investigated the effect of nobiletin on bacterial lipopolysaccharide (LPS)-induced expression of tissue factor (TF), a trigger protein for the blood coagulation cascade, and studied the possible mechanism of TF transcriptional regulation. THP-1 monocytic cells stimulated with LPS showed an increased expression of both TF protein and mRNA levels. However, pretreatment with nobiletin resulted in inhibition of LPS-induced expression of both TF protein and mRNA in a dose-dependent manner. Electrophoretic mobility shift assays revealed that binding of nuclear proteins from LPS-stimulated THP-1 cells to the NF-κB or AP-1 binding motif was increased as compared to non-stimulated control cells. Such increased binding activities were significantly reduced by pretreatment with nobiletin. Binding activity of nuclear proteins to the Sp1 binding motif was observed irrespective of LPS stimulation, but Sp1 activation was inhibited by nobiletin treatment of the cells. Treatment of THP-1 cells with Sp1-specific small interfering RNA (Sp1 siRNA) abolished the ability of LPS to induce TF activity. A similar reduction in the level of TF mRNA was also observed upon treatment of cells with Sp1 siRNA. These studies reveal that constitutive Sp1 activation is an essential event for transcriptional activation of TF, and nobiletin prevents LPS-induced TF expression by inhibiting NF-κB, AP-1, and Sp1 activation.

Keywords: Nobiletin; Tissue factor; LPS; NF-κB; AP-1; Sp1

The phosphatidylinositol 3-kinase/Akt pathway negatively regulates Nod2-mediated NF-κB pathway by Ling Zhao; Joo Y. Lee; Daniel H. Hwang (pp. 1515-1525).

Nucleotide-binding oligomerization domain containing proteins (Nods) are intracellular pattern recognition receptors (PRRs) that recognize conserved moieties of bacterial peptidoglycan and activate downstream signaling pathways, including NF-κB pathway. Here, we show that Nod2 agonist muramyldipeptide (MDP) induces Akt phosphorylation in time and dose-dependent manner. The pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) (wortmannin) and dominant-negative forms of p85 (the regulatory subunit of PI3K) or Akt enhance, while constitutive active forms of p110 (the catalytic subunit of PI3K) or Akt inhibit, NF-κB activation and the target gene interleukin (IL)-8 induced by MDP. In addition, the pharmacological inhibitors of PI3K (wortmannin and LY294002) enhance phosphorylation of NF-κB p65 on Ser529 and Ser536 residues, which result in enhanced p65 transactivation activity. Furthermore, we show that the inhibition of PI3K by the pharmacological inhibitors prevent the inactivation of glycogen synthase kinase (GSK)-3β, suggesting that the negative regulation of PI3K/Akt on MDP-induced NF-κB activation is at least in part mediated through inactivation of GSK-3β. Taken together, our results demonstrate that PI3K/Akt pathway is activated by Nod2 agonist MDP and negatively regulates NF-κB pathway downstream of Nod2 activation. Our results suggest that PI3K/Akt pathway may involve in the resolution of inflammatory responses induced by Nod2 activation.

Keywords: Abbreviations; TLRs; Toll-like receptors; PRR; pattern recognition receptors; LRR; leucine-rich repeat domain; Nod; the nucleotide-binding oligomerization domain family; NF-κB; nuclear factor-κB; JNK; c-Jun N-terminal kinase; MAPK; mitogen-activated protein kinase; PGN; peptidoglycan; LPS; lipopolysaccharide; iE-DAP; γ-; d; -glutamyl-meso-diaminopimelic acid; MDP; muramyldipeptide MurNAc-; l; -Ala-; d; -isoGln; GSK-3β; glycogen synthase kinase-3β; RICK; Rip-like interacting CLARP kinase; IKK; IκB kinase; IκB; inhibitor of NF-κB; DN; dominant-negative; CD; Crohn's diseaseNod2; PI3K; NF-κB; GSK-3β; Inflammation; Resolution

Inhibition of 6-hydroxydopamine-induced oxidative damage by 4,5-dihydro-3 H-2-benzazepine N-oxides by Ramón Soto-Otero; Estefanía Méndez-Álvarez; Sofía Sánchez-Iglesias; Fedor I. Zubkov; Leonid G. Voskressensky; Alexey V. Varlamov; Modesto de Candia; Cosimo Altomare (pp. 1526-1537).

A number of new analogs of 3,3-dimethyl-4,5-dihydro-3 H-2-benzazepine 2-oxide, structurally related to the nitrone spin trap α-phenyl- N- tert-butylnitrone (PBN), were synthesized and evaluated for their activity in vitro as protectants against oxidative stress induced in rat brain mitochondria by 6-hydroxydopamine (6-OHDA), a neurotoxin producing experimental model of Parkinson's disease (PD). As assessed by a fluorimetric assay, all 2-benzazepine-based nitrones were shown to decrease hydroxyl radicals (OH) generated during 6-OHDA autoxidation. The inhibition effects on theOH formation shown by the 5- gem-dimethyl derivatives, 2–4 times higher than those of the corresponding 5-methyl derivatives, were attributed to the flattening effect of the 5- gem-dimethyl group on the azepine ring, which should enhance nitrone reactivity and/or increase stability of the radical adducts. In contrast, owing to steric hindrance, a methyl group to C-1 diminishes theOH-scavenging activity of the nitrone group. All the assayed compounds were more potent than PBN as inhibitors of 6-OHDA-induced lipid peroxidation (LPO) and protein carbonylation (PCO), taken as an indicator of mitochondrial protein oxidative damage. The most promising antioxidant (compound11), bearing 5- gem-dimethyl and spiro C-3 cyclohexyl groups, highlighted in this study as the best features, inhibited LPO and PCO with IC₅₀ values of 20 and 48μM, respectively, showing a potency improvement over PBN of two order magnitude. Both LPO and PCO inhibition potency data were found primarily related to theOH-scavenging activities, whereas lipophilicity plays a role in improving the LPO (but not PCO) inhibition, as a statistically valuable two-parameter equation proved.

Keywords: Abbreviations; PD; Parkinson's disease; AD; Alzheimer's disease; ALS; amyotrophic lateral sclerosis; 6-OHDA; 6-hydroxydopamine; MPTP; N; -methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PBN; α-phenyl-; N; -; tert; -butylnitrone; ROS; reactive oxygen species; OH; hydroxyl radical; LPO; lipid peroxidation; RCS; reactive carbonyl species; PCO; protein carbonylation; THA; terephthalic acid; TBA; thiobarbituric acid; TBARS; thiobarbituric acid reactive substances; TCA; trichloroacetic acid; MDA; malondialdehyde; MAO; monoamine oxidase; BSA; bovine serum albumin; SDS; sodium dodecylsulfate2-Benzazepine nitrones; Neuroprotection; Radical scavengers; 6-Hydroxydopamine; Lipid peroxidation; Protein carbonylation

Relative contribution of rat cytochrome P450 isoforms to the metabolism of caffeine: The pathway and concentration dependence by Marta Kot; Władysława A. Daniel (pp. 1538-1549).

The aim of the present study was to estimate the relative contribution of rat P450 isoforms to the metabolism of caffeine and to assess the usefulness of caffeine as a marker substance for estimating the activity of P450 in rat liver and its potential for pharmacokinetic interactions in pharmacological experiments. The results obtained using rat cDNA-expressed P450s indicated that 8-hydroxylation was the main oxidation pathway of caffeine (70%) in the rat. CYP1A2 was found to be a key enzyme catalyzing 8-hydroxylation (72%) and substantially contributing to 3- N-demethylation (47%) and 1- N-demethylation (37.5%) at a caffeine concentration of 0.1mM (relevant to “the maximum therapeutic concentration in humans”). Furthermore, CYP2C11 considerably contributed to 3- N-demethylation (31%). The CYP2C subfamily (66%) – mainly CYP2C6 (27%) and CYP2C11 (29%) – played a major role in catalyzing 7- N-demethylation. At higher substrate concentrations, the contribution of CYP1A2 to the metabolism of caffeine decreased in favor of CYP2C11 ( N-demethylations) and CYP3A2 (mainly 8-hydroxylation). The obtained results were confirmed with liver microsomes (inhibition and correlation studies). Therefore, caffeine may be used as a marker substance for assessing the activity of CYP1A2 in rats, using 8-hydroxylation (but not 3- N-demethylation—like in humans); moreover, caffeine may also be used to simultaneously, preliminarily estimate the activity of CYP2C using 7- N-demethylation as a marker reaction. Hence caffeine pharmacokinetics in rats may be changed by drugs affecting the activity of CYP1A2 and/or CYP2C, e.g. by some antidepressants.

Keywords: Abbreviations; CYP; P450; cytochrome P450; FMO; flavin-containing monooxygenase; HPLC; high performance liquid chromatography; αNF; α-naphthoflavone; SULF; sulfaphenazole; CIM; cimetidine; QUIN; quinine; trans; -DCE; trans; -1,2-dichloroethylene; ERY; erythromycin; K; _m; the Michaelis constant; V; _max; maximum velocity of the reactionCaffeine metabolism; Rat cytochrome P450; cDNA-expressed isoforms; Liver microsomes; Cytochrome P450 inhibitors; Correlation analysis

Mouse N-acetyltransferase type 2, the homologue of human N-acetyltransferase type 1 by Akane Kawamura; Isaac Westwood; Larissa Wakefield; Hilary Long; Naixia Zhang; Kylie Walters; Christina Redfield; Edith Sim (pp. 1550-1560).

There is increasing evidence that human arylamine N-acetyltransferase type 1 (NAT1, EC 2.3.1.5), although first identified as a homologue of a drug-metabolising enzyme, appears to be a marker in human oestrogen receptor positive breast cancer. Mouse Nat2 is the mouse equivalent of human NAT1. The development of mouse models of breast cancer is important, and it is essential to explore the biological role of mouse Nat2. We have therefore produced mouse Nat2 as a recombinant protein and have investigated its substrate specificity profile in comparison with human NAT1. In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids. We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2. The steroid analogue, bisphenol A, also inhibits mouse Nat2 enzymic activity and is shown by NMR spectroscopy, through shifts in proton peaks, to bind close to the active site. A three-dimensional structure for human NAT1 has recently been released, and we have used this crystal structure to generate a model of the mouse Nat2 structure. We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.

Keywords: Arylamine; N; -acetyltransferase/NAT; Enzyme inhibition; Selective estrogen receptor modulator/SERM; Steroid; Xenobiotic; Breast cancer

Effects of oximes on rate of decarbamylation of human red blood cell AChE measured with two different methods by Saskia Eckert; Peter Eyer; Marco Melzer; H. Thiermann; Franz Worek (pp. 1561-1566).

Treatment regimen of poisonings by organophosphorus (OP) compounds usually includes oxime therapy. The treatment options in soman poisoning are very limited due to rapid aging of the inhibited acetylcholinesterase (AChE), when the enzyme species is considered as irreversibly inhibited and resistant towards reactivation by oximes. Hence, oxime treatment probably comes too late in realistic scenarios. As an alternative, protecting part of the enzyme by reversible inhibition prior to soman exposure has been proposed. One means of protecting against soman poisoning is the prophylactic use of certain reversible inhibitors (carbamates) of AChE. The question whether there is a possibility of an interaction between pre-treating carbamates and oximes at AChE arises.Therefore we studied the effects of the oximes obidoxime, HI 6 and MMB-4 on the rate of decarbamylation for physostigmine- and pyridostigmine-inhibited human erythrocyte AChE both in a dynamically working in vitro model and a static cuvette system.Our results show that HI 6 increased the rate of decarbamylation for both physostigmine- and pyridostigmine-inhibited enzyme in both systems, the observed effect by HI 6 increasing with higher doses. Obidoxime had a slightly accelerating effect on the pyridostigmine-inhibited enzyme. MMB-4 applied to pyridostigmine-inhibited AChE in the static system only showed no difference to the experiments made in absence of oxime. No oxime showed a tendency to retard the rate of decarbamylation.

Keywords: Abbreviations; AChE; acetylcholinesterase (E.C.3.1.1.7); AU; absorbance units; DTNB; 5,5′-dithiobis(2-nitrobenzoic acid); HI 6; 1-[[[4-(aminocarbonyl)-pyridinio]methoxy]methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride; MMB-4; N; ,; N; ′-monomethylenebis(4-pyridiniumaldoxime)dibromide; Obi(obidoxime); 1,1′-(oxybis-methylene)bis[4-(hydroxyimino)methyl]pyridinium dichloride; Pyr; pyridostigmineCarbamate prophylaxis; Acetylcholinesterase; Pyridostigmine; Physostigmine; Decarbamylation; Oximes

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Biochemical Pharmacology (v.75, #7)