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Biochemical Pharmacology (v.69, #8)

Editorial Advisory Board (pp. iii).

Target identification and validation in drug discovery: the role of proteomics by Karla K. Kopec; Donna Bozyczko-Coyne; Michael Williams (pp. 1133-1139).

Proteomics, the study of cellular protein expression, is an evolving technology platform that has the potential to identify novel proteins involved in key biological processes in the cell that may serve as potential drug targets. While proteomics has considerable theoretical promise, individual cells/tissues have the potential to generate many millions of proteins while the current analytical technologies that involve the use of time-consuming two dimensional gel electrophoresis (2DIGE) and various mass spectrometry (MS) techniques are unable to handle complex biological samples without multiple high-resolution purification steps to reduce their complexity. This can significantly limit the speed of data generation and replication and requires the use of bioinformatic algorithms to reconstitute the parent proteome, a process that does not always result in a reproducible outcome. In addition, membrane bound proteins, e.g., receptors and ion channels, that are the targets of many existing drugs, are not amenable to study due, in part, to limitations in current proteomic techniques and also to these being present in low abundance and thus disproportionally represented in proteome profiles. Subproteomes with reduced complexity have been used to generate data related to specific, hypothesis-driven questions regarding target identification, protein-interaction networks and signaling pathways. However progress to date, with the exception of diagnostic proteomics in the field of cancer, has been exceedingly slow with an inability to put such studies in the context of a larger proteome, limiting the value of the information. Additionally the pathway for target validation (which can be more accurately described at the preclinical level as target confidence building) remains unclear. It is important that the ability to measure and interrogate proteomes matches expectations, avoiding a repetition of the disappointment and subsequent skepticism that accompanied what proved to be unrealistic expectations for the rapid contribution of data based on the genome maps, to biomedical research.

Keywords: Proteomics; Drug discovery; Target validation; Target confidence building

Stimulation of CD95-induced apoptosis in T-cells by a subtype specific neutral sphingomyelinase inhibitor by Eberhard Amtmann; Margot Z�ller (pp. 1141-1148).

Neutral sphingomyelinase (nSMase) has been supposed to be involved in the activation of anti-apoptotic genes and, thus, could well sustain autoimmune reactions by preventing activation induced death of autoreactive T-cells. When screening cellular extracts for SMase activity in the range between pH 6.5 and 8.5 various murine tissue samples as well as cell lines of murine and human origin displayed peaks of activity, both, at pH 7.0 and 8.0. In contrast, T-cells (human T-cell lymphoma and PHA stimulated murine lymph node cells) and monocytic leukemia cells were lacking SMase activity at pH 8.0. Only one peak of activity was found at pH 7.0. Recently we described an inhibitory compound, C11AG which selectively suppresses nSMase activity. In dose–response assays using cellular extracts the pH 7.0 nSMase turned out to be almost 100-fold more sensitive to the inhibitor than the pH 8.0 nSMase. In Jurkat T-cell lymphoma cells lacking the pH 8.0 nSMase, treatment with C11AG enhanced sensitivity to apoptosis: the concentration of CD95-specific antibody anti-APO1 could be lowered by six-fold in order to induce cell death. Concomitantly the expression of the anti-apoptotic protein A1 was found to be down-regulated. In the joints of arthritic mice, apoptosis of T-cells was stimulated after application of C11AG. Accordingly, C11AG displayed curative effects on experimental arthritis: swelling and inflammation were found to be significantly alleviated.

Keywords: Neutral sphingomyelinase; Sphingomyelinase inhibitors; Apoptosis; Autoimmune diseases; Arthritis; Undecylideneaminoguanidine

Exploitation of the unusual thermodynamic properties of human myeloperoxidase in inhibitor design by Walter Jantschko; Paul Georg Furtm�ller; Martina Zederbauer; Karin Neugschwandtner; Isabella Lehner; Christa Jakopitsch; J�rgen Arnhold; Christian Obinger (pp. 1149-1157).

Myeloperoxidase plays a fundamental role in oxidant production by neutrophils. It uses hydrogen peroxide and chloride to catalyze the production of hypochlorous acid (HOCl), which contributes to both bacterial killing and oxidative injury of host tissue. Thus, MPO is an interesting target for anti-inflammatory therapy. Here, based on the extraordinary and MPO-specific redox properties of its intermediates compound I and compound II, we present a rational approach in selection and design of reversible inhibitors of HOCl production mediated by MPO. In detail, indole and tryptamine derivatives were investigated for their ability to reduce compounds I and II and to affect the chlorinating activity of MPO. It is shown that these aromatic one-electron donors bound to the hydrophobic pocket at the distal heme cavity and were oxidized efficiently by compound I ( k₃), which has a one-electron reduction potential of 1.35V. By contrast, compound II ( E°′ of the compound II/ferric couple is 0.97V) reduction ( k₄) was extremely slow. As a consequence compound II, which does not participate in the halogenation cycle, accumulated. The extent of chlorinating activity inhibition (IC₅₀) was related to the k₃/ k₄ ratio. The most efficient inhibitors were 5-fluorotryptamine and 5-chlorotryptamine with IC₅₀ of 0.79μM and 0.73μM and k₃/ k₄ ratios of 386,000 and 224,000, respectively. The reversible mechanism of inhibition is discussed with respect to the enzymology of MPO and the development of drugs against HOCl-dependent tissue damage.

Keywords: Abbreviations; MPO; myeloperoxidase; LPO; lactoperoxidase; HRP; horseradish peroxidase; E; °′; standard reduction potential; MCD; monochlorodimedonMyeloperoxidase; Chlorination activity; Inflammation; Tissue damage; Reduction potential; Inhibitor

Hydrogen peroxide degradation and selective carbidopa-induced cytotoxicity against human tumor lines by Judith A. Gilbert; Linda M. Frederick; Lori J. Pobst; Matthew M. Ames (pp. 1159-1166).

The carcinoid tumor, an uncommon neuroendocrine neoplasm, is associated with serotonin overproduction as is more common small cell lung carcinoma (SCLC). α-Methyl-dopahydrazine (carbidopa), an inhibitor of the serotonin synthetic enzyme aromatic-l-amino acid decarboxylase, proved lethal to NCI-H727 lung carcinoid cells as well as NCI-H146 and NCI-H209 SCLC cells, but not to five other human tumor cell lines of differing origins [Gilbert JA, Frederick LM, Ames MM. The aromatic-l-amino acid decarboxylase inhibitor carbidopa is selectively cytotoxic to human pulmonary carcinoid and small cell lung carcinoma cells. Clin. Cancer Res. 2000;6:4365–72]. The mechanism of carbidopa cytotoxicity remained an unanswered question. We present data here that incubation of the catechol carbidopa (100μM) in RPMI and DMEM culture media yielded molar equivalents of hydrogen peroxide (H₂O₂) within 2–4h. Alkaline elution studies revealed carbidopa-dependent single-strand DNA breaks in sensitive carcinoid cells comparable to those induced by similar concentrations of H₂O₂. Neither compound induced significant DNA damage in carbidopa-resistant NCI-H460 large cell lung carcinoma cells. Furthermore, when carbidopa was incubated with a variety of tumor cell types, not only were decreased media H₂O₂ concentrations detected in the presence of cells, but cell lines least sensitive to carbidopa degraded exogenous H₂O₂ more rapidly than did sensitive cells. Implicated in these studies, pyruvate degraded H₂O₂ in RPMI in a dose- and time-dependent manner and reversed carbidopa-induced cytotoxicity to carcinoid cells. Extracellular pyruvate levels produced per h by resistant large cell lung carcinoma cells averaged four-fold that of sensitive carcinoid cells plated at equal density (24h time course). Finally, carbidopa exposure (100μM, 24h) depleted extracellular pyruvate from sensitive carcinoid cells, but reduced pyruvate levels from resistant NCI-H460 cells less than 17%.

Keywords: Abbreviations; TPH; tryptophan hydroxylase; AAAD; aromatic-; l; -amino acid decarboxylase; carbidopa; α-methyl-dopahydrazine; SCLC; small cell lung carcinoma; H; ₂; O; ₂; hydrogen peroxide; ROS; reactive oxygen speciesJEL classification; Molecular and Cellular PharmacologyHuman; Carcinoid; SCLC; Hydrogen peroxide; Pyruvate

Necrotic and apoptotic features of cell death in response to Foscan^� photosensitization of HT29 monolayer and multicell spheroids by Sophie Marchal; Anas Fadloun; Estelle Maugain; Marie-Ange D’Hallewin; François Guillemin; Lina Bezdetnaya (pp. 1167-1176).

Photodynamic therapy (PDT) is an approved anticancer treatment modality that eliminates unwanted cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which is selectively taken up by tumor cells. Present study reports the modalities of cell death after photosensitization of human adenocarcinoma HT29 monolayer and spheroid cells with a second generation photosensitizer Foscan^®. Kinetics of apoptosis and necrosis after Foscan^®-PDT in monolayer cells determined by flow cytometry using labeling of cleaved poly(ADP-ribose) polymerase (PARP) and staining with propidium iodide (PI) demonstrated that Foscan^® was not a strong inducer of apoptosis and necrosis was a prevailing mode of cell death. Cytochrome c release (cyt c) and mitochondrial membrane potential (Δ ψ_m) addressed by flow cytometry technique at different time points post-Foscan^®-PDT demonstrated that cell photoinactivation was governed by these mitochondrial events. Foscan^®-loaded HT29 multicell spheroids, subjected to irradiation with different fluence rates and equivalent light doses, displayed much better tumoricidal activity at the lowest fluence rate used. Apoptosis, measured by caspase-3 activation was evidenced only in spheroids irradiated with the lowest fluence rate and moderate fluence inducing 65% of cell death. Application of higher fluence rates for the same level of photocytotoxicity did not result in caspase-3 activation. The observation of the fluence rate-dependent modulation of caspase-3 activity in spheroids offers the possibility of regulating the mechanism of direct cell photodamage and could be of great potential in the clinical context.

Keywords: Abbreviations; cyt; c; cytochrome; c; ER; endoplasmic reticulum; LD; lethal dose; Δ; ψ; _m; mitochondrial membrane potential; PARP; poly(ADP-ribose) polymerase; PDT; photodynamic therapy; PI; propidium iodidePhotodynamic therapy; Foscan; ^®; Apoptosis; Mitochondrial events; Caspase-3; Spheroids

2-Aminoethoxydiphenyl borate perturbs hormone-sensitive calcium stores and blocks store-operated calcium influx pathways independent of cytoskeletal disruption in human A549 lung cancer cells by Shanthala Padar; Diptiman D. Bose; John C. Livesey; David W. Thomas (pp. 1177-1186).

Recent studies have identified novel actions for 2-aminoethoxydiphenyl borate (2-APB) in triggering calcium release and enhancing calcium influx induced by the depletion of intracellular calcium stores. In this study, we have examined the effects of 2-APB on the human lung adenocarcinoma A549 cell line, which we have previously shown displays a unique calcium influx response, when ER calcium stores are depleted by thapsigargin (TG) treatment. Here, we show that low concentrations of 2-APB failed to induce the rapid augmentation of TG-activated calcium influx previously reported for other cell types. We observed that store-operated calcium (SOC) channels in the A549 cell line exhibited short-term sensitivity to low doses of 2-APB, perhaps reflecting a delayed augmentation of SOC channel activity or the recruitment of 2-APB-insensitive SOC channels. In both intact and permeabilized cells, 2-APB effectively discharged a subset of A549 calcium pools corresponding to the hormone-sensitive intracellular calcium stores. The 2-APB-induced calcium release produced a long-lasting perturbation of the adenosine triphosphate (ATP)-releasable calcium pools, effectively uncoupling ATP-activated calcium release even, when stores are replenished with calcium. In contrast to previous reports, we found that disruption of either the actin or microtubule-based cytoskeleton failed to block the 2-APB-induced effects on calcium signaling in A549 cells. Our study describes novel cytoskeletal-independent effects of 2-APB on Ca²⁺-signaling pathways, revealing differentially sensitive Ca²⁺-influx pathways and long-term perturbation of hormone-sensitive Ca²⁺ stores.

Keywords: Abbreviations; IP; ₃; inositol 1,4,5-trisphosphate; IP; ₃; R; inositol 1,4,5-trisphosphate receptor; SOC; store-operated Ca; ²⁺; channel; 2-APB; 2-aminoethoxydiphenyl borate; ER; endoplasmic reticulum; PM; plasma mebrane; SERCA; sarcoplasmic/endoplasmic reticulum Ca; ²⁺; -ATPase; CCCP; carbonyl cyanide 3-chlorophenylhydrazone; TG; thapsigargin; [Ca; ²⁺; ]; _i; intracellular Ca; ²⁺; HBSS; Hank's balanced salt solution2-Aminoethoxydiphenyl borate; Store-operated calcium influx; Calcium stores; A549 lung cells; Inositol 1,4,5-trisphosphate receptor; Cytoskeleton

A spectrophotometric assay for fatty acid amide hydrolase suitable for high-throughput screening by Paul A. De Bank; David A. Kendall; Stephen P.H. Alexander (pp. 1187-1193).

Signalling via the endocannabinoids anandamide and 2-arachidonylglycerol appears to be terminated largely through the action of the enzyme fatty acid amide hydrolase (FAAH). In this report, we describe a simple spectrophotometric assay to detect FAAH activity in vitro using the ability of the enzyme to hydrolyze oleamide and measuring the resultant production of ammonia with a NADH/NAD⁺-coupled enzyme reaction. This dual-enzyme assay was used to determine K_m and V_max values of 104μM and 5.7nmol/min/mgprotein, respectively, for rat liver FAAH-catalyzed oleamide hydrolysis. Inhibitor potency was determined with the resultant rank order of methyl arachidonyl fluorophosphonate>phenylmethylsulphonyl fluoride>anandamide. This assay system was also adapted for use in microtiter plates and its ability to detect a known inhibitor of FAAH demonstrated, highlighting its potential for use in high-throughput screening.

Keywords: Abbreviations; CPF; crude particulate fraction; FAAH; fatty acid amide hydrolase; GDH; glutamate dehydrogenase; MAFP; methyl arachidonyl fluorophosphonate; PMSF; phenylmethylsulphonyl fluorophosphonateFatty acid amide hydrolase; Assay; Spectrophotometer; Enzyme; Inhibition; Cannabinoid

Increased Akt protein expression is associated with decreased ceramide content in skeletal muscle of troglitazone-treated mice by Anna Planavila; Marta Alegret; Rosa M. S�nchez; Ricardo Rodr�guez-Calvo; Juan Carlos Laguna; Manuel V�zquez-Carrera (pp. 1195-1204).

Although it is generally believed that thiazolidinediones ameliorate insulin resistance by lowering circulating free fatty acids, direct effects of these drugs in skeletal muscle may also contribute to their antidiabetic action. We report that troglitazone administration to mice for 1 day increased the protein expression of Akt (two-fold induction, P<0.001) in skeletal muscle without significant changes in the levels of free fatty acids in plasma. Increased Akt protein expression was associated with reduced phospho-AMP-activated protein kinase abundance and with a fall in the phosphorylation of acetyl-CoA carboxylase, which in turn resulted in an increase in the content of muscular malonyl-CoA (2.4-fold, P<0.05) and lactate (1.4-fold, P<0.05). Troglitazone treatment did not affect the mRNA levels of either Akt1 or Akt2, suggesting that a transcriptional mechanism was not involved, but caused a dramatic reduction in the content of muscular ceramides (76%, P<0.001), lipid-derived second messengers known to increase Akt degradation. Our data indicate that troglitazone treatment inhibited de novo ceramide synthesis, since the content of its precursor, palmitoyl-CoA, was reduced (55%, P=0.05). These results were confirmed in C2C12 myotubes, where troglitazone treatment increased Akt protein expression and prevented the reduction of this protein and the increase in ceramide levels caused by palmitate. These findings implicate ceramide as an important intermediate in the regulation of Akt after troglitazone treatment.

Keywords: Abbreviations; ACC; acetyl-CoA carboxylase; AMPK; AMP-activated protein kinase; CTE; cytosolic acyl-CoA thioesterase; M-CPT-I; muscle-type carnitine palmitoyl-transferase; PGC-1; PPARγ coactivator 1; PPAR; peroxisome proliferator-activated receptorAkt; PPAR; Troglitazone; AMPK; Malonyl-CoA; Ceramide

Curcumin-induced histone hypoacetylation: The role of reactive oxygen species by Jiuhong Kang; Jie Chen; Yufeng Shi; Jie Jia; Yuntao Zhang (pp. 1205-1213).

Curcumin (Cur), a well-known dietary pigment derived from Curcuma longa, is a promising anticancer drug, but its in vivo target molecules remain to be clarified. Here we report that exposure of human hepatoma cells to Cur led to a significant decrease of histone acetylation. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are the enzymes controlling the state of histone acetylation in vivo. Cur treatment resulted in a comparable inhibition of histone acetylation in the absence or presence of trichostatin A (the specific HDAC inhibitor), and showed no effect on the in vitro activity of HDAC. In contrast, the domain negative of p300 (a most potent HAT protein) could block the inhibition of Cur on histone acetylation; and the Cur treatment significantly inhibited the HAT activity both in vivo and in vitro. Thus, it is HAT, but not HDAC that is involved in Cur-induced histone hypoacetylation. At the same time, exposure of cells to low or high concentrations of Cur diminished or enhanced the ROS generation, respectively. And the promotion of ROS was obviously involved in Cur-induced histone hypoacetylation, since Cur-caused histone acetylation and HAT activity decrease could be markedly diminished by the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) or their combination, but not by their heat-inactivated forms. The data presented here prove that HAT is one of the in vivo target molecules of Cur; through inhibiting its activity, Cur induces histone hypoacetylation in vivo, where the ROS generation plays an important role. Considering the critical roles of histone acetylation in eukaryotic gene transcription and the involvement of histone hypoacetylation in the lose of cell viability caused by high concentrations of Cur, these results open a new door for us to further understand the molecular mechanism involved in the in vivo function of Cur.

Keywords: Abbreviations; Cur; curcumin; HDAC; histone deacetylase; HAT; histone acetyltransferase; ROS; reactive oxygen species; TSA; tricostatin A; SOD; superoxide dismutase; CAT; catalase; p300 DN; p300 domain negative; CHX; cycloheximideCurcumin; Histone acetylation; Histone acetyltransferase (HAT); Histone deacetylase (HDAC); Reactive oxygen species (ROS); p300

Involvement of uracil nucleotides in protection of cardiomyocytes from hypoxic stress by Smadar Yitzhaki; Vladimir Shneyvays; Kenneth A. Jacobson; Asher Shainberg (pp. 1215-1223).

Cardiomyocytes express one or more subtypes of P2 receptors for extracellular nucleotides. P2 purinoceptors, which are activated by nucleotides, are classified as P2X or P2Y: P2X receptors are ligand-gated intrinsic ion channels, and P2Y receptors are G protein-coupled receptors. Extracellular pyrimidine and purine nucleotides are released from the heart during hypoxia. Although the cardioprotective effects of purines acting via purinoceptors were studied intensively, the physiological role of uracil nucleotide-responsive P2Y₂, P2Y₄, P2Y₆, and P2Y₁₄ receptors is still unclear, especially in the cardiovascular system. This study revealed that uridine-5′-triphosphate (UTP) protected cultured rat cardiomyocytes during hypoxia and explored the UTP signaling pathway leading to this cardioprotection. We found that UTP, but not UDP or uridine, significantly reduced cardiomyocyte death induced by hypoxia. Incubation with UTP for 1h, before exposure to hypoxic conditions, protected the cells 24h later. The cardioprotective effect of UTP was reduced in the presence of the P2 antagonist suramin. In addition, UTP caused a transient increase of [Ca²⁺]_i in cardiomyocytes. Pyridoxal-5′-phosphate-6-azophenyl-2,4-disulfonate (PPADS) or Reactive blue 2 (RB-2), other antagonists of P2 receptors, abolished the [Ca²⁺]_i elevation caused by UTP. We used various inhibitors of the Ca²⁺ signaling pathway to show that UTP elevated levels of [Ca²⁺]_i, originating from intracellular sources, via activation of phospholipase C and the IP₃ receptor. Interestingly, these inhibitors of the Ca²⁺ signaling pathway did not prevent the immediate protective effect caused by UTP. Although mitochondrial K_ATP channels are involved in other preconditioning mediator pathways, the involvement of these channels in the cardioprotective effect induced by UTP was ruled out, because 5-hydroxydecanoic acid (5-HD), a specific inhibitor of these channels, did not prevent the protection.

Keywords: Abbreviations; 2APB; 2-aminoethoxydiphenylborane; ATP; adenosine 5′-triphosphate; FITC; fluorescein isothiocyanate; 5-HD; 5-hydroxydecanoic acid; IP; ₃; inositol 1,4,5-trisphosphate; LDH; lactate dehydrogenase; PBS; phosphate buffered saline; PC; preconditioning; PI3K; phosphoinositide-3-kinase; PLC; phospholipase C; PKC; protein kinase C; PPADS; pyridoxal-5′-phosphate-6-azophenyl-2,4-disulfonate; RB-2; Reactive blue 2; U73122; (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione); UDP; uridine-5′-diphosphate; UTP; uridine-5′-triphosphateP2Y; ₂; nucleotide receptor; G protein-coupled receptor; Pyrimidines; Cardioprotection; Ischemia; Preconditioning

Stimulation of intracellular Ca²⁺ elevation in neutrophils by thiol-oxidizing phenylarsine oxide by Jih-Pyang Wang; Jaw-Ji Tsai; Yu-San Chen; Mei-Feng Hsu (pp. 1225-1234).

Phenylarsine oxide (PAO), a trivalent arsenical compound, stimulated [Ca²⁺]_i elevation in rat neutrophils in a Ca²⁺-containing medium but caused no appreciable response in a Ca²⁺-free medium. PAO also induced external Mn²⁺ entry, which was inhibited by N-acetyl-l-cysteine (NAC), but failed to elicit any appreciable Ba²⁺ and Sr²⁺ entry. Pretreatment of neutrophils with thiol-reducing agents including dithiothreitol (DTT), NAC, 2,3-dimercapto-1-propanol (DMP), 2,3-dimercaptopropane-1-sulfonic acid (DMPS) and tris-(2-carboxyethyl)phosphine (TCEP), all greatly inhibited PAO-induced [Ca²⁺]_i elevation. Addition of Ni²⁺ or La³⁺ followed by PAO stimulation also attenuated the Ca²⁺ signals in a concentration-dependent manner. PAO had no significant effect on the production of reactive oxygen intermediates (ROI) and nitric oxide (NO) nor did it decrease cellular low molecular weight thiols levels. PAO-induced [Ca²⁺]_i elevation was significantly inhibited by 1-[6-[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1 H-pyrrole-2,5-dione (U-73122), the inhibitor of phospholipase C-coupled processes, genistein, a general tyrosine kinase inhibitor, phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, calyculin A, a cortical actin stabilizer, 2-(4-morpholinyl)-8-phenyl-4 H-1-benzopyran-4-one (LY 294002), a phosphoinositide 3-kinase inhibitor, 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1 H-imidazole (SKF-96365), and cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A), the blockers of receptor-gated and store-operated Ca²⁺ channels, whereas there was no appreciable effect exerted by aristolochic acid, a phospholipase A₂ inhibitor, 7-nitroindazole and N-(3-aminomethyl)benzylacetamidine (1400W), the blockers of NO synthase, and by suspension in a Na⁺-deprived medium. In contrast, 2-aminoethoxydiphenyl borane (2-APB), the blocker of IP₃ receptor and Ca²⁺ influx, enhanced the PAO-induced response. PAO had no effect on the plasma membrane Ca²⁺-ATPase (PMCA) activity in the pharmacological isolated neutrophil preparation and the neutrophil membrane fractions. These results indicate that PAO stimulates [Ca²⁺]_i rise in rat neutrophils mainly through the oxidation of vicinal thiol groups on the cell surface membrane to activation of a non-store operated Ca²⁺ entry (non-SOCE) without affecting the activity of PMCA and the plasmalemmal Na⁺/Ca²⁺ exchanger.

Keywords: Abbreviations; 1400W; N; -(3-aminomethyl)benzylacetamidine; AA; arachidonic acid; 2-APB; 2-aminoethoxydiphenyl borane; CPA; cyclopiazonic acid; DAF-2/DA; 4,5-diaminofluorescein diacetate; DMP; 2,3-dimercapto-1-propanol; DMPS; 2,3-dimercaptopropane-1-sulfonic acid; DTT; dithiothreitol; GEA3162; 5-amino-3-(3,4-dichlorophenyl)1,2,3,4-oxatriazolium; HBSS; Hanks’ balanced salt solution; H; ₂; DCF/DA; 2′,7′-dichlorodihydrofluorescein diacetate; IP; ₃; d; -; myo; -inositol 1,4,5-trisphosphate; LY 294002; 2-(4-morpholinyl)-8-phenyl-4; H; -1-benzopyran-4-one; mBBr; monobromobimane; MDL-12,330A; cis; -; N; -(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine; NAC; N; -acetyl-; l; -cysteine; NO; nitric oxide; PAO; phenylarsine oxide; PIK3; phosphoinositde-3-kinase; PKC; protein kinase C; PLC; phospholipase C; PMA; phorbol 12-myristate 13-acetate; PMCA; plasma membrane Ca; ²⁺; -ATPase; ROI; reactive oxygen intermediates; SERCA; sarco/endoplasmic reticulum Ca; ²⁺; -ATPase; SKF-96365; 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1; H; -imidazole; SOCE; store-operated Ca; ²⁺; entry; TCEP; tris-(2-carboxyethyl)phosphine; U-73122; 1-[6-[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1; H; -pyrrole-2,5-dionePhenylarsine oxide; Cation entry; Intracellular free-Ca; ²⁺; Signal transduction; Non-store operated Ca; ²⁺; entry; Neutrophils

Cys^2,7EtαCGRP is a potent agonist for CGRP₁ receptors in SK-N-MC cells by Christina Nodin; Georges Vauquelin; Bengt von Mentzer (pp. 1235-1240).

The present study reveals that cystein^2,7 ethyl-amideαCGRP (Cys^2,7EtαCGRP), an advertised calcitonin gene-related peptide 2 (CGRP₂) receptor subtype-selective agonist, is also a potent agonist for the calcitonin gene-related peptide 1 (CGRP₁) receptors natively expressed in the SK-N-MC human neuroblastoma cell line. Cys^2,7EtαCGRP and α calcitonin gene-related peptide (αCGRP) promote cyclic AMP accumulation in intact SK-N-MC cells to the same extent with EC₅₀ of 1.6±0.2 and 0.4±0.08nM, respectively. The antagonist α calcitonin gene-related peptide-8-37 (αCGRP-(8-37)) produces a concentration-dependent rightward shift of the αCGRP- and Cys^2,7EtαCGRP concentration–response curves with K_B-values (71±33 and 47±21nM, respectively). The competitive antagonism by αCGRP-(8-37) and the similar K_B-values suggests that αCGRP and Cys^2,7EtαCGRP stimulate the same receptor. In competition binding studies with [¹²⁵I]-αCGRP on SK-N-MC cell membranes, Cys^2,7EtαCGRP and αCGRP-(8-37) display high affinity for the majority of the binding sites with K_i-values of 0.030±0.013 and 0.60±0.013nM, respectively. The present findings are at odds with the proclaimed utilization of Cys^2,7EtαCGRP as a CGRP₂ receptor-selective pharmacological tool. Differences between the agonistic profile of this ligand in this and other experimental systems might be species – or even cell type – dependent.

Keywords: Abbreviations; αCGRP; α calcitonin gene-related peptide; cAMP; cyclic AMP; αCGRP-(8-37); α calcitonin gene-related peptide-8-37; Cys; ^2,7; EtαCGRP; cystein2,7 ethyl-amideαCGRP; Cys; ^2,7; AcmαCGRP; cystein2,7 acetyl-methylαCGRP; CGRP; ₁; calcitonin gene-related peptide 1; CGRP; ₂; calcitonin gene-related peptide 2; CRLR; calcitonin receptor-like receptor protein; RAMP; receptor activity modifying proteinCalcitonin gene-related peptide; Cys; ^2,7; Et calcitonin gene-related peptide; CGRP-(8-37); CGRP receptor subtypes; Receptor activity modifying protein

Cyclooxygenation of the arachidonoyl side chain of 1-arachidonoylglycerol and related compounds block their ability to prevent anandamide and 2-oleoylglycerol metabolism by rat brain in vitro by Christopher J. Fowler; Gunnar Tiger (pp. 1241-1245).

In the present study, the abilities of cyclooxygenated derivatives of 1-arachidonoylglycerol and related compounds to prevent the metabolism of [³H]2-oleoylglycerol and [³H]anandamide by cytosolic and membrane fractions, respectively, have been investigated. For each compound, nine concentrations (range 0.2–100μM) were tested. 1-Arachidonoylglycerol inhibited the hydrolysis of [³H]2-oleoylglycerol with a p I₅₀ value of 5.17±0.04 (maximum attainable inhibition 88%). In contrast, the 1-glyceryl esters of prostaglandin D₂, E₂ and F_2α were very weak inhibitors of this hydrolysis. Similarly, prostaglandin D₂, prostaglandin D₂ ethanolamide and prostaglandin D₂ serinol amide produced <20% inhibition of [³H]2-oleoylglycerol metabolism at any concentration tested, in contrast to previous data with arachidonic acid, anandamide and arachidonoyl serinol which are all able to inhibit metabolism of this substrate under the assay conditions used here. A similar pattern was seen for all the compounds with respect to the inhibition of [³H]anandamide hydrolysis by the membrane fractions. Thus, cyclooxygenation of the arachidonoyl side chain greatly reduces the ability of 1-arachidonoylglycerol and related compounds to inhibit the hydrolysis of [³H]2-oleoylglycerol and [³H]anandamide.

Keywords: Abbreviations; AEA; anandamide; 2-AG; 2-arachidonoylglycerol; 1-AG; 1-arachidonoylglycerol; FAAH; fatty acid amide hydrolase; MAGL; monoacylglycerol lipase; 2-OG; 2-oleoylglycerolMonoacylglycerol lipase; Fatty acid amide hydrolase; Anandamide; 2-Arachidonoylglycerol; Endocannabinoid; Cyclooxygenase

Gα_q potentiation of adenylate cyclase type 9 activity through a Ca²⁺/calmodulin-dependent pathway by Medhane G. Cumbay; Val J. Watts (pp. 1247-1256).

Adenylate cyclase (EC 4.6.1.1) type 9 (AC9) activity has been shown to be inhibited by PMA activation of novel protein kinase C (nPKC) isoforms. In the current study the effect on AC9 activity of activating PKC in physiological relevant manner was examined. Contrary to the anticipated inhibitory effect of activating PKCs through G_q-coupled receptors, activation of transiently expressed G_q-coupled serotonin 5-HT_2A or muscarinic M₅ receptors resulted in the potentiation of isoproterenol-stimulated cyclic AMP accumulation in HEK293 cells stably expressing AC9 (HEK-AC9). Consistent with G_q-mediated activation of PKC, the addition of the PKC inhibitor bisindolylmaleimide further potentiated isoproterenol-stimulated cyclic AMP accumulation. Expression of a constitutively active mutant of Gα_q in HEK-AC9 cells also produced an enhancement in basal and isoproterenol-stimulated cyclic AMP accumulation. We also examined the role of Gα_q-mediated release of intracellular Ca²⁺ on the observed potentiation of AC9 activity, by depleting intracellular Ca²⁺ stores with thapsigargin. In Ca²⁺-depleted HEK-AC9 cells, activation of transiently expressed M₅ receptors resulted in inhibition of isoproterenol-stimulated cyclic AMP accumulation that was blocked by bisindolylmaleimide, indicating that M₅ potentiation of AC9 activity requires Ca²⁺. This prompted us to examine the effects of the calmodulin antagonist W7 and the Ca²⁺/calmodulin-dependent kinase II (CaMK II) inhibitor KN-93. Pretreating cells with W7 and KN-93 significantly inhibited M₅-mediated potentiation of isoproterenol-stimulated cyclic AMP accumulation in HEK-AC9 cells, suggesting that Gα_q potentiation of AC9 activity involves Ca²⁺/calmodulin and CaMK II. This data provides evidence for Ca²⁺-mediated potentiation of AC9 activity.

Keywords: Adenylate cyclase; Protein kinase C; Calmodulin; G alpha q; Learning and memory; Muscarinic receptor

cGMP (guanosine 3′,5′-cyclic monophosphate) transport across human erythrocyte membranes by Chung-Pu Wu; Hannah Woodcock; Stephen B. Hladky; Margery A. Barrand (pp. 1257-1262).

Human erythrocytes produce cGMP that can be eliminated by phosphodiesterases or active efflux transporters. The efflux can be studied under controlled conditions as ATP-dependent uptake into inside-out membrane vesicles. However, widely differing values for the transport rates have been reported. We have here examined factors that influence the uptake rates measured and thus may explain these discrepancies. Both the ionic composition of the buffer used during uptake and the mode of vesicle preparation were found to affect the observed transport rates. Furthermore it was apparent that different blood donors expressed on their erythrocytes different amounts of both MRP4 and MRP5, transporters that have been putatively linked to cGMP efflux across erythrocyte membranes. These differences in expression were reflected in differences in rates of cGMP uptake into inside-out erythrocyte membrane vesicles. Calculations based on the transport rates observed using vesicles suggest that efflux may be the principal means for eliminating cGMP from human erythrocytes.

Keywords: Abbreviations; OSV; one-step inside-out membrane vesicles prepared by the spontaneous vesiculation method; SKNV; inside-out membrane vesicles prepared by the modified Steck and Kant; Nycodenz methodErythrocyte; cGMP; ABC transporter; Multidrug-resistance associated protein; Membrane vesicles; MRP4

Induction of apoptosis and modulation of production of reactive oxygen species in human endothelial cells by diphenyleneiodonium by Aneta Balcerczyk; Mirosław Soszynski; Dorota Rybaczek; Tomasz Przygodzki; Agata Karowicz-Bilinska; Janusz Maszewski; Grzegorz Bartosz (pp. 1263-1273).

Diphenyleneiodonium (DPI) inhibits activity of flavoenzymes like NAD(P)H oxidase, the major source of superoxide anion in cardiovascular system, but affects also other oxidoreductases. Contradictory data have been published concerning the effect of diphenyleneiodonium on the production of reactive oxygen species in cells, both inhibitory and stimulatory action of DPI being reported. We have examined the effect of DPI on the cellular production of reactive oxygen and nitrogen species (ROS/RNS) and on the proliferation and apoptosis of human vascular endothelial cells. We found increased oxidation of ROS-sensitive probes (dihydrorhodamine 123 and 2′,7′-dichlorodihydrofluorescein diacetate) when DPI (20μM–100μM) was present in the treated cells. However, oxidation of the fluorogenic probes was inhibited if DPI (20μM–100μM) was removed from the reaction medium after cell preincubation. These results suggest an artifactual oxidation of the fluorogenic probes by DPI or its metabolites. A similar pattern of influence of DPI on the production of NO (measured with 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate) was observed. Modulation of generation of reactive oxygen and nitrogen species in DPI-treated cells influenced the nitration of tyrosine residues of cellular proteins, estimated by Western blotting. Decreased level of nitration generally paralleled the lowered production of ROS. A decreased 3-(4,5-dimethylthiazolyl)-3-3(4-sulphophenyl) tetrazolium (MTT) reducing activity of cells for was observed immediately after 1h treatment of human endothelial cells with DPI (1μM–100μM), in spite of lack of changes in cell viability estimated by other methods. These results point to a next limitation of MTT in estimation of viability of cells treated with oxidoreductase inhibitors. DPI inhibited the proliferation of HUVECs as well as immortalized cell line HUVEC-ST, as assessed by acid phosphatase activity test and measurement of total nucleic acid content. Proapoptotic action of DPI was observed 12h after incubation with this compound.

Keywords: Abbreviations; DAF-FM; 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate; dHR 123; dihydrorhodamine 123; DPI; diphenyleneiodonium; H; ₂; DCF-DA; 2′,7′-dichlorodihydrofluorescein diacetate; JC-1; 1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide; MTT; 3-(4,5-dimethylthiazolyl)-3-3(4-sulphophenyl) tetrazolium; RNS; reactive nitogen species; ROS; reactive oxygen speciesDiphenyleneiodonium; Apoptosis; Reactive oxygen/nitrogen species; Endothelial cells

A downstream role for protein kinase Cα in the cytosolic phospholipase A₂-dependent protective signalling mediated by peroxynitrite in U937 cells by Andrea Guidarelli; Liana Cerioni; Ilaria Tommasini; Bernhard Br�ne; Orazio Cantoni (pp. 1275-1286).

Exposure to an otherwise non-toxic concentration of peroxynitrite (ONOO⁻) promotes toxicity in U937 cells supplemented with pharmacological inhibitors of protein kinase C (PKC). This effect is not associated with enhanced formation of H₂O₂ and is in fact causally linked to inhibition of the cytoprotective signalling driven by endogenous arachidonic acid (AA). The outcome of various approaches using PKC or phospholipase A₂ inhibitors, cytosolic phospholipase A₂ or PKCα antisense-oligonucleotide-transfected cells and supplementation with exogenous AA or tetradecanoylphorbol acetate, as well as PKC down-regulated cells, indicated that ONOO⁻ promotes AA-dependent cytosol to membrane translocation of PKCα, an event critical for the cytoprotective signalling under investigation. Evidence for a similar mechanism regulating survival of human monocytes exposed to ONOO⁻ is also presented. These results, along with our previous work on this topic, contribute to the definition of the mechanism whereby monocytes survive to ONOO⁻ in inflamed tissues.

Keywords: JEL classification; ToxicologyAbbreviations; AS-ONs; antisense-oligonucleotides; AA; arachidonic acid; AACOCF; ₃; arachidonyl trifluoromethyl ketone; calcein-AM; calcein acetoxymethyl ester; CsA; cyclosporin A; cPLA; ₂; cytosolic phospholipase A; ₂; DHR; dihydrorhodamine 123; ETYA; 5,8,11,14-eicosatetraynoic acid; MPT; mitochondrial permeability transition; NS-ONs; nonsense-oligonucleotides; PLA; ₂; phospholipase A; ₂; PKC; protein kinase C; TPA; tetradecanoylphorbol acetatePeroxynitrite; Protein kinase Cα; Cytosolic phospholipase A; ₂; Arachidonic acid; Mitochondrial permeability transition; Cell death

Dexamethasone-induced methylprednisolone hemisuccinate hydrolase: Its identification as a member of the rat carboxylesterase 2 family and its unique existence in plasma by Tomomi Furihata; Masakiyo Hosokawa; Ayako Fujii; Maher Derbel; Tetsuo Satoh; Kan Chiba (pp. 1287-1297).

Carboxylesterases (CESs) play important roles in the metabolism of many ester-drugs. In the present study, we identified and characterized dexamethasone-induced methylprednisolone hemisuccinate (MPHS) hydrolase in rat liver microsomes. Intraperitoneal injection of dexamethasone resulted in a significant increase in the level of MPHS hydrolase activity accompanied by induction of a specific CES isozyme. Since the biochemical characteristics of the induced CES isozyme were very similar to those of rat CES RL4, we hypothesized that these were the same enzymes. The results of nano-electrospray ionization tandem mass spectrometry analysis revealed that both dexamethasone-induced CES isozyme and CES RL4 possessed identical peptide fragments to those ofAB010635, a rat CES2 isozyme, supporting our hypothesis. Furthermore, the results of reverse transcription-polymerase chain reaction showed that the amount ofAB010635 mRNA in dexamethasone-treated liver was greater than that in control liver. To confirm thatAB010635 encodes dexamethasone-induced CES isozyme, cDNA cloning was performed and the obtained cDNA was expressed in Sf9 cells by using a baculovirus-mediated expression system. The recombinant CES protein could hydrolyze MPHS and exhibited biochemical characteristics similar to those of CES RL4. Collectively, the results indicated that dexamethasone-induced MPHS hydrolase in liver microsomes is a rat CES2 isozyme. Interestingly, the results also showed that this rat CES2 isozyme exists in plasma and that the amount of this protein is increased by dexamethasone. These findings, together with the findings described above, provide important information for the study of phramacokinetics and pharmacodynamics of ester-drugs as well as for the study of CESs.

Keywords: Abbreviations; CES; carboxylesterase; CES RH1; rat carboxylesterase RH1; CES RL1; rat carboxylesterase RL1; CES RL4; rat carboxylesterase RL4; Dex; dexamethasone; ESI/MS/MS; electrospray ionization tandem mass spectrometory; MPHS; methylprednisolone hemisuccinate; Ms; microsomes; PNPA; p; -nitrophenylacetate; SDS-PAGE; sodium dodecyl sulphate-polyacrylamide gel electrophoresisCarboxylesterase; Dexamethasone; Methylprednisolone hemisuccinate; Induction; Baculovirus-mediated expression system; Microsomes

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Biochemical Pharmacology (v.69, #8)