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Biochemical Pharmacology (v.72, #6)

Editorial Advisory Board (pp. iii).

Toxin insights into nicotinic acetylcholine receptors by Sébastien Dutertre; Richard J. Lewis (pp. 661-670).

Venomous species have evolved cocktails of bioactive peptides to facilitate prey capture. Given their often exquisite potency and target selectivity, venom peptides provide unique biochemical tools for probing the function of membrane proteins at the molecular level. In the field of the nicotinic acetylcholine receptors (nAChRs), the subtype specific snake α-neurotoxins and cone snail α-conotoxins have been widely used to probe receptor structure and function in native tissues and recombinant systems. However, only recently has it been possible to generate an accurate molecular view of these nAChR–toxin interactions. Crystal structures of AChBP, a homologue of the nAChR ligand binding domain, have now been solved in complex with α-cobratoxin, α-conotoxin PnIA and α-conotoxin ImI. The orientation of all three toxins in the ACh binding site confirms many of the predictions obtained from mutagenesis and docking simulations on homology models of mammalian nAChR. The precise understanding of the molecular determinants of these complexes is expected to contribute to the development of more selective nAChR modulators. In this commentary, we review the structural data on nAChR–toxin interactions and discuss their implications for the design of novel ligands acting at the nAChR.

Keywords: Abbreviations; AChBP; acetylcholine binding protein; CAPS; 3-(cyclohexylamino)-1-propanesulfonic acid; Cbtx; α-cobratoxin; PnIA; α-conotoxin PnIA; ImI; α-conotoxin ImI; MLA; methyllycaconitine; PEG; polyethylene glycol; EPI; epibatidine; LOB; α-lobeline; nAChR; nicotinic acetylcholine receptorNicotinic acetylcholine receptor (nAChR); Homology models; Docking simulations; X-ray protein structure; Conotoxins; Structure–activity relationships (SAR)

Oxidative stress by ascorbate/menadione association kills K562 human chronic myelogenous leukaemia cells and inhibits its tumour growth in nude mice by Julien Verrax; Julie Stockis; Aurélie Tison; Henryk S. Taper; Pedro Buc Calderon (pp. 671-680).

The effect of oxidative stress induced by the ascorbate/menadione-redox association was examined in K562 cells, a human erythromyeloid leukaemia cell line. Our results show that ascorbate enhances menadione redox cycling, leading to the formation of intracellular reactive oxygen species (as shown by dihydrorhodamine 123 oxidation). The incubation of cells in the presence of both ascorbate/menadione and aminotriazole, a catalase inhibitor, resulted in a strong decrease of cell survival, reinforcing the role of H₂O₂ as the main oxidizing agent killing K562 cells. This cell death was not caspase-3-dependent. Indeed, neither procaspase-3 and PARP were processed and only a weak cytochrome c release was observed. Moreover, we observed only 23% of cells with depolarized mitochondria. In ascorbate/menadione-treated cells, DNA fragmentation was observed without any sign of chromatin condensation (DAPI and TUNEL tests). The cell demise by ascorbate/menadione is consistent with a necrosis-like cell death confirmed by both cytometric profile of annexin-V/propidium iodide labeled cells and by light microscopy examination. Finally, we showed that a single i.p. administration of the association of ascorbate and menadione is able to inhibit the growth of K562 cells by about 60% (in both tumour size and volume) in an immune-deficient mice model. Taken together, these results reinforced our previous claims about a potential application of the ascorbate/menadione association in cancer therapy.

Keywords: Ascorbate; Menadione; Oxidative stress; Cancer; Apoptosis; necrosis

Apigenin-induced-apoptosis is mediated by the activation of PKCδ and caspases in leukemia cells by Melissa A. Vargo; Oliver H. Voss; Frantisek Poustka; Arturo J. Cardounel; Erich Grotewold; Andrea I. Doseff (pp. 681-692).

Apigenin, a flavone abundantly found in fruits and vegetables, exhibits antiproliferative, anti-inflammatory, and antimetastatic activities through poorly defined mechanisms. In the present study, the treatment of different cell lines with apigenin resulted in selective antiproliferative and apoptotic effect in monocytic and lymphocytic leukemias. Apigenin-induced-apoptosis was mediated by the activation of caspase-9 and caspase-3. Apigenin was found intracellularly and localized to the mitochondria. Treatment of monocytic cells with apigenin was accompanied by an increase in reactive oxygen species (ROS) and phosphorylation of the MAPKs, p38 and ERK. However, the inhibition of ROS, p38 or ERK failed to block apoptosis, suggesting that these cellular responses induced by apigenin are not essential for the induction of apoptosis. In addition, apigenin induced the activation of PKCδ. Pharmacological inhibition of PKCδ, the expression of dominant-negative PKCδ and silencing of PKCδ in leukemia cells showed that apigenin-induced-apoptosis requires PKCδ activity. Together, these results indicate that this flavonoid provides selective activity to promote caspase-dependent-apoptosis of leukemia cells and uncover an essential role of PKCδ during the induction of apoptosis by apigenin.

Keywords: Apigenin-induced-apoptosis; PKC; Caspase; Leukemia

Different accumulation of cisplatin, oxaliplatin and JM216 in sensitive and cisplatin-resistant human cervical tumour cells by Laura Martelli; Francesco Di Mario; Eugenio Ragazzi; Piero Apostoli; Roberto Leone; Paola Perego; Guido Fumagalli (pp. 693-700).

The significance of reduced drug accumulation in resistance to cisplatin was investigated by using cisplatin, oxaliplatin and JM216 (hydrophobicity rank: JM216>oxaliplatin>cisplatin) in human squamous cell carcinoma cell line A431 and its cisplatin-resistant counterpart A431/Pt. While cisplatin showed a resistance factor of 2.6, oxaliplatin and JM216 circumvented the resistance. Platinum accumulation after cisplatin exposure was lower (2.4-fold) in A431/Pt than in A431 cells, whereas a similar accumulation was found in the two cell lines when oxaliplatin or JM216 were used, thereby suggesting the capability of the latter drugs to bypass the accumulation defect. In the A431 cell line platinum accumulated to a similar extent after exposure to cisplatin, oxaliplatin or JM216, while in A431/Pt cells, Platinum accumulation depended on the hydrophobicity of the drug, and an increased hydrophobicity favours the uptake. No difference in efflux of cisplatin was found between the two cell lines. The values of platinum–DNA binding in A431 cells were similar for cisplatin and JM216 and higher than those of oxaliplatin. In A431/Pt cells: (i) Pt–DNA binding levels of JM216 remained as in sensitive ones; (ii) Pt–DNA levels of cisplatin and oxaliplatin were very similar and nearly two-fold lower than those of JM216. Such results, in this cell system characterized by a low level of cisplatin resistance, support a model whereby platinum uptake occurs by a mechanism of facilitated diffusion, perhaps involving a gated channel, which can be lost during the selection of the drug-resistant variant(s). The hydrophobicity of the drug can be the key to bypass resistance.

Keywords: A431 cervix squamous cell carcinoma; Platinum drugs; resistance; accumulation; Pt–DNA binding; hydrophobicity

Histone deacetylase inhibitors and transforming growth factor-β induce 15-hydroxyprostaglandin dehydrogenase expression in human lung adenocarcinoma cells by Min Tong; Yunfei Ding; Hsin-Hsiung Tai (pp. 701-709).

Histone deacetylase (HDAC) inhibitors have been actively exploited as potential anticancer agents. To identify gene targets of HDAC inhibitors, we found that HDAC inhibitors such as sodium butyrate, scriptaid, apicidin and oxamflatin induced the expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a potential cyclooxygenase-2 (COX-2) antagonist and tumor suppressor, in a time and concentration dependent manner in A549 and H1435 lung adenocarcinoma cells. Detailed analyses indicated that HDAC inhibitors activated the 15-PGDH promoter-luciferase reporter construct in transfected A549 cells. A representative HDAC inhibitor, scriptaid, and its negative structural analog control, nullscript, were further evaluated at the chromatin level. Scriptaid but not nullscript induced a significant accumulation of acetylated histones H3 and H4 which were associated with the 15-PGDH promoter as determined by chromatin immunoprecipitation assay. Transforming growth factor-β1 (TGF-β1) also induced the expression of 15-PGDH in a time and concentration dependent manner in A549 and H1435 cells. Induction of 15-PGDH expression by TGF-β1 was synergistically stimulated by the addition of Wnt3A which was inactive by itself. However, combination of TGF-β and an HDAC inhibitor, scriptaid, only resulted in an additive effect. Together, our results indicate that 15-PGDH is one of the target genes that HDAC inhibitors and TGF-β may induce to exhibit tumor suppressive effects.

Keywords: 15-PGDH; COX-2; HDAC Inhibitors; TGF-β; Wnt; Tumor suppressor

The diabetogenic antibiotic streptozotocin modifies the tryptic digest pattern for peptides of the enzyme O-GlcNAc-selective N-acetyl-β-d-glucosaminidase that contain amino acid residues essential for enzymatic activity by Thomas N. Lee; William E. Alborn; Michael D. Knierman; Robert J. Konrad (pp. 710-718).

Streptozotocin (STZ) inhibits O-GlcNAc-selective N-acetyl-β-d-glucosaminidase ( O-GlcNAcase), the enzyme that removes O-GlcNAc from proteins. The active site of the enzyme was recently proposed to include aspartates 174, 175, and 177, with STZ inhibition via a transition state analog. We explored the effect of STZ on the tryptic peptide digest pattern of O-GlcNAcase. LC/MS/MS analysis demonstrated that STZ modified two areas of the enzyme. One peptide, LGCFEIAK (894–901), in a C-terminal region previously proposed to possess O-GlcNAcase activity, was methylated by STZ. Another peptide, EYEIEFIYIASPGLDITFSNPK (128–149), was detected only after treatment with STZ and was in an N-terminal region, overlapping a glutamate-rich area containing an adjacent phenylalanine residue. No covalent modification of this peptide could be demonstrated. Detection of this peptide after treatment with STZ was accompanied by the simultaneous inability to detect the nearby peptide KLDQVSQFGCR (157–167), which contains a cysteine residue recently shown to be essential for enzymatic activity. To determine which of the first two peptides might also be important for O-GlcNAcase activity, site-specific mutagenesis was performed. Mutation of the N-terminal phenylalanine and serine residues resulted in almost complete inhibition of activity. In contrast, mutation of conserved C-terminal glycine and cysteine residues caused little inhibition of enzymatic activity. Together, these data extend the region of the active site N-terminally and give independent evidence to support the idea that STZ inhibits O-GlcNAcase through formation of a transition state analog that resides in the active site of the enzyme and in doing so alters its conformation and ensuing tryptic digest pattern.

Keywords: Abbreviations; PBS; phosphate buffered saline; STZ; streptozotocin; O; -GlcNAcase; O; -GlcNAc-selective; N; -acetyl-β-; d; -glucosaminidase; OGT; O; -GlcNAc transferase; GlcNAc; N; -acetylglucosamine; MNU; methylnitrosourea; PUGNAc; O; -(2-acetamido-2-deoxy-; d; -glucopyranosylide) amino-; N; -phenylcarbamateβ-Cells; Streptozotocin; O; -GlcNAcase; OGT; Islets

Evaluation of the antidiabetic activity of DPP IV resistant N-terminally modified versus mid-chain acylated analogues of glucose-dependent insulinotropic polypeptide by Nigel Irwin; Gillian C. Clarke; Brian D. Green; Brett Greer; Patrick Harriott; Victor A. Gault; Finbarr P.M. O’Harte; Peter R. Flatt (pp. 719-728).

Glucose dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone with therapeutic potential for type 2 diabetes due to its insulin-releasing and antihyperglycaemic actions. However, development of GIP-based therapies is limited by N-terminal degradation by DPP IV resulting in a very short circulating half-life. Numerous GIP analogues have now been generated exhibiting DPP IV resistance and extended bioactivity profiles. In this study, we report a direct comparison of the long-term antidiabetic actions of three such GIP molecules, N-AcGIP, GIP(Lys³⁷PAL) and N-AcGIP(Lys³⁷PAL) in obese diabetic ( ob/ ob) mice. An extended duration of action of each GIP analogue was demonstrated prior to examining the effects of once daily injections (25nmolkg⁻¹ body weight) over a 14-day period. Administration of either N-AcGIP, GIP(Lys³⁷PAL) or N-AcGIP(Lys³⁷PAL) significantly decreased non-fasting plasma glucose and improved glucose tolerance compared to saline treated controls. All three analogues significantly enhanced glucose and nutrient-induced insulin release, and improved insulin sensitivity. The metabolic and insulin secretory responses to native GIP were also enhanced in 14-day analogue treated mice, revealing no evidence of GIP-receptor desensitization. These effects were accompanied by significantly enhanced pancreatic insulin following N-AcGIP(Lys³⁷PAL) and increased islet number and islet size in all three groups. Body weight, food intake and circulating glucagon were unchanged. These data demonstrate the therapeutic potential of once daily injection of enzyme resistant GIP analogues and indicate that N-AcGIP is equally as effective as related palmitate derivatised analogues of GIP.

Keywords: Abbreviations; GIP; glucose-dependent insulinotropic polypeptide; GLP-1; glucagon-like peptide-1; DPP IV; dipeptidylpeptidase IV; (; ob; /; ob; ) mice; obese diabetic mice; HPLC; high performance liquid chromatography; MALDI-TOF; matrix-assisted laser desorption ionization-time of flight; AUC; area under curve; Lys; lysine; PAL; palmitateGlucose-dependent insulinotropic polypeptide; ob; /; ob; mice; Glucose homeostasis; Analogues; Type 2 diabetes

Expression of transporters potentially involved in the targeting of cytostatic bile acid derivatives to colon cancer and polyps by Maria R. Ballestero; Maria J. Monte; Oscar Briz; Felipe Jimenez; Francisco Gonzalez-San Martin; Jose J.G. Marin (pp. 729-738).

Drug targeting might help to overcome resistance to chemotherapy. Here we investigated whether colon cancer and polyps do express functional carriers involved in the uptake of cytostatic bile acid derivatives, in this case Bamet-UD2 [ cis-diammine-bisursodeoxycholate-platinum(II)], which has been reported to be taken up by colon cancer cells “in vitro�?, efficiently induce apoptosis and overcome resistance to cisplatin. Although at lower levels than in ileum, a detectable expression of ASBT, OATP8/1B3, OCT1 and OSTα in colon tissue was found, which was not impaired in colon cancer or polyps. The expression of OATP-A/1A2 and OSTβ was also found in colon, but this was markedly decreased in neoplastic colon tissue. In contrast, the expression of OATP-C/1B1 was low in colon but significantly enhanced in neoplastic colon tissue. MDR1 and MRP2 were poorly expressed in colon as compared with ileum, whereas MRP3 expression was higher in colon than in ileum. The abundance of mRNA for these ABC proteins was not changed in colon cancer or polyps. When RNA from different tissues was injected to Xenopus laevis oocytes their ability to take up taurocholate and Bamet-UD2 was enhanced (healthy ileum>healthy colon≈neoplastic colon tissue). In all cases, uptake was lower for taurocholate than for Bamet-UD2, probably due to that ASBT mediates sodium-dependent uptake of both substrates, whereas additional transporters expressed in these tissues can participate in Bamet-UD2 uptake. In conclusion, our results suggest that the use of cytostatic bile acid derivatives might be a good pharmacological strategy for the treatment of colon tumors.

Keywords: Abbreviations; ASBT; apical sodium-dependent bile salt transporter; MDR; multidrug resistance protein; MRP; multidrug resistance-associated protein; OATP; organic anion transporting polypeptide; OCT; organic cation transporter; OST; organic solute transporter; TC; taurocholateASBT; Chemotherapy; Gut; Intestine; Resistance; Tumor

Diverse signalling by different chemokines through the chemokine receptor CCR5 by Anja Mueller; Nasir G. Mahmoud; Philip G. Strange (pp. 739-748).

We have investigated the signalling properties of the chemokine receptor, CCR5, using several assays for agonism: stimulation of changes in intracellular Ca²⁺ or CCR5 internalisation in CHO cells expressing CCR5 or stimulation of [³⁵S]GTPγS binding in membranes of CHO cells expressing CCR5.Four isoforms of the chemokine CCL3 with different amino termini (CCL3, CCL3(2–70), CCL3(5–70), CCL3L1) were tested in these assays in order to probe structure/activity relationships. Each isoform exhibited agonism. The pattern of agonism (potency, maximal effect) was different in the three assays, although the rank order was the same with CCL3L1 being the most potent and efficacious. The data show that the amino terminus of the chemokine is important for signalling. A proline at position 2 (CCL3L1) provides for high potency and efficacy but the isoform with a serine at position 2 (CCL3(2–70)) is as efficacious in some assays showing that the proline is not the only determinant of high efficacy.We also increased the sensitivity of CCR5 signalling by treating cells with sodium butyrate, thus increasing the receptor/G protein ratio. This allowed the detection of a change in intracellular Ca²⁺ after treatment with CCL7 and Met-RANTES showing that these ligands possess measurable but low efficacy.This study therefore shows that sodium butyrate treatment increases the sensitivity of signalling assays and enables the detection of efficacy in ligands previously considered as antagonists. The use of different assay systems, therefore, provides different estimates of efficacy for some ligands at this receptor.

Keywords: Abbreviations; CHO cells; Chinese hamster ovary cells; ECL; enhanced chemiluminescence; FCS; foetal calf serum; FITC; fluorescein isothiocyanate; GPCR; G protein-coupled receptor; HIV; human immunodeficiency virus; MCP; monocyte chemoattractant protein; MIP; macrophage inflammatory protein; PBS; phosphate-buffered saline; RANTES; regulated on activation, normal T cell expressed and secretedChemokine receptor CCR5; Chemokines; [; ³⁵; S]GTPγS binding; Ca; ²⁺; mobilisation; Receptor internalisation

Effective attenuation of acute lung injury in vivo and the formyl peptide-induced neutrophil activation in vitro by CYL-26z through the phosphoinositide 3-kinase γ pathway by Yu-Hsiang Kuan; Ruey-Hseng Lin; Yeh-Long Chen; Lo-Ti Tsao; Cherng-Chyi Tzeng; Jih-Pyang Wang (pp. 749-760).

5-[4-Acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one (CYL-26z) inhibited the polymorphonuclear leukocyte (PMNL) infiltration and protein leakage into the lungs in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice as determined on the basis of PMNL and protein contents in bronchoalveolar lavage (BAL) fluid and myeloperoxidase (MPO) content in whole lung extracts. CYL-26z also attenuated the formyl-Met-Leu-Phe (fMLP)-induced neutrophil chemotaxis and respiratory burst in vitro (IC₅₀ 8.4±0.9μM and 2.0±0.6μM, respectively). CYL-26z had no effect on superoxide anion (O₂⁻) generation during dihydroxyfumaric acid autoxidation or on the NADPH oxidase activity in two cell-free systems (the arachidonic acid-induced assembly of NADPH oxidase and the preassembled oxidase caused by phorbol ester treatment), whereas it inhibited NaF-induced respiratory burst. Inhibition of respiratory burst by CYL-26z was readily reversible by washing. Only slight, but significant, inhibition of extracellular signal regulated kinase (ERK) phosphorylation and p38 mitogen-activated protein kinase (MAPK) activation in response to fMLP by CYL-26z up to 30μM was obtained. CYL-26z effectively blocked the formation of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5) P₃) as determined by immunofluorescence microscopy and flow cytometry assays and the dual phosphorylation of protein kinase B (PKB/Akt) on S473 and T308 residues in fMLP-stimulated neutrophils. The membrane recruitment of p110γ and Ras, the Ras activation, and the association between p110γ and Ras were also attenuated by CYL-26z. These results indicate that the blockade of Ras activation by CYL-26z attenuated the downstream phosphoinositide 3-kinase (PI3K) γ signaling, which is involved in chemoattractant-induced neutrophil chemotaxis and respiratory burst, and may have a beneficial anti-inflammatory effect on ALI.

Keywords: Abbreviations; ALI; acute lung injury; BAL; bronchoalveolar lavage; CYL-26z; 5-[4-acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one; ERK; extracellular signal regulated kinase; fMLP; formyl-Met-Leu-Phe; HBSS; Hanks’ balanced salt solution; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; MAPKAPK-2; MAPK-activated protein kinase-2; MPO; myeloperoxidase; O; ₂; ⁻; superoxide anion; PDK; 3′-phosphoinositide-dependent kinase; PI3K; phosphoinositide 3-kinase; PKB/Akt; protein kinase B; PMA; phorbol 12-myristate 13-acetate; PtdIns(3,4,5); P; ₃; phosphatidylinositol-3,4,5-trisphosphateCYL-26z; Acute lung injury; Neutrophils; Chemotaxis; Respiratory burst; Ras; PI3Kγ

Involvement of hydrogen peroxide in the differentiation and apoptosis of preosteoclastic cells exposed to arsenite by K.H. Szymczyk; B.A.E. Kerr; T.A. Freeman; C.S. Adams; M.J. Steinbeck (pp. 761-769).

Long-term exposure to sodium arsenite (AsO₂) promotes the development of various cancers. Paradoxically, arsenic also induces pro-myelomonocytic leukemia cell differentiation, and at higher concentrations, apoptosis. The present study investigated the effects of AsO₂ on preosteoclasts. When treated with 2.5–5μM AsO₂, RAW264.7 cells underwent osteoclast differentiation as evidenced by an increase in the number of multinucleate cells expressing tartrate resistant acid phosphatase (TRAP). The appearance of these phenotypic markers was preceded by a low level increase in extracellular production of H₂O₂ and was prevented by the addition of catalase (4.5μg/ml), an enzyme that removes H₂O₂. Only at high concentrations (10–25μM) of AsO₂ was a significant loss of cell viability and a high level increase in H₂O₂ production (1.5μM) observed. Apoptosis was blocked by pretreatment with diphenylene iodonium chloride (2μM), a NAD(P)H-flavoprotein inhibitor, suggesting the involvement of NADPH-oxidase. The data show that AsO₂, dose-dependently, stimulates increasing amounts of H₂O₂ production. Moreover, at concentrations found in tissues of individuals exposed to geochemical AsO₂, osteoclasts underwent an H₂O₂-dependent differentiation. Therefore, chronic exposure to low-level amounts of AsO₂ could result in increased bone resorption and contribute to bone related pathologies.

Keywords: Abbreviations; AsO; ₂; sodium arsenite; DPI; diphenylene iodonium chloride; ERK; extracellular signal-regulated protein kinases; JNK; Jun N-terminal kinase; RANKL; ligand for receptor activator of nuclear factor-κB; RANK; receptor activator of NF-κB; PMA; 4β-phorbol-12-myristate-13-acetate; ROS; reactive oxygen species; TRAP; tartrate resistant acid phosphatase; TUNEL; terminal deoxynucleotidyl transferase biotin-dUTP nick end labelingArsenite; Hydrogen peroxide; Osteoclasts; Apoptosis; Tartrate-resistant acid phosphatase (TRAP); Caspase-3

Pharmacological characterization of recombinant N-type calcium channel (Ca_v2.2) mediated calcium mobilization using FLIPR by Elfrida R. Benjamin; Farhana Pruthi; Shakira Olanrewaju; Shen Shan; Denise Hanway; Xuesong Liu; Rok Cerne; Daniel Lavery; Kenneth J. Valenzano; Richard M. Woodward; Victor I. Ilyin (pp. 770-782).

The N-type voltage-gated calcium channel (Ca_v2.2) functions in neurons to regulate neurotransmitter release. It comprises a clinically relevant target for chronic pain. We have validated a calcium mobilization approach to assessing Ca_v2.2 pharmacology in two stable Ca_v2.2 cell lines: α1_B, α2δ, β₃-HEK-293 and α1_B, β₃-HEK-293. Ca_v2.2 channels were opened by addition of KCl and Ca²⁺ mobilization was measured by Fluo-4 fluorescence on a fluorescence imaging plate reader (FLIPR⁹⁶). Ca_v2.2 expression and biophysics were confirmed by patch-clamp electrophysiology (EP). Both cell lines responded to KCl with adequate signal-to-background. Signals from both cell lines were inhibited by ω-conotoxin (ctx)-MVIIa and ω-conotoxin (ctx)-GVIa with IC₅₀ values of 1.8 and 1nM, respectively, for the three-subunit stable, and 0.9 and 0.6nM, respectively, for the two-subunit stable. Other known Ca_v2.2 blockers were characterized including cadmium, flunarizine, fluspirilene, and mibefradil. IC₅₀ values correlated with literature EP-derived values. Novel Ca_v2.2 pharmacology was identified in classes of compounds with other primary pharmacological activities, including Na⁺ channel inhibitors and antidepressants. Novel Na⁺ channel compounds with high potency at Ca_v2.2 were identified in the phenoxyphenyl pyridine, phenoxyphenyl pyrazole, and other classes. The highest potency at Ca_v2.2 tricyclic antidepressant identified was desipramine.

Keywords: Abbreviations; Ctx-GVIa; ω-conotoxin GVIa; Ctx-MVIIa; ω-conotoxin MVIIa; EP; electrophysiology; FLIPR; fluorescence imaging plate reader; HEK-293; human embryonic kidney 293; Ca; _v; 1; L-type voltage-gated calcium channel; Ca; _v; 2.2; N-type voltage-gated calcium channel; PBSC; 4-(4-fluorophenoxy)benzaldehyde semicarbazoneN-type calcium channel; Calcium mobilization; Pharmacology; Sodium channel inhibitor; Antidepressant; Fluorescence imaging plate reader

Clozapine bioactivation induces dose-dependent, drug-specific toxicity of human bone marrow stromal cells: A potential in vitro system for the study of agranulocytosis by Avril Pereira; Brian Dean (pp. 783-793).

Clozapine, an atypical antipsychotic drug effective in treatment of refractory schizophrenia causes potentially life-threatening agranulocytosis. The drug undergoes bioactivation to a toxic, chemically reactive intermediate with capacity to target stromal cells, central components of the bone marrow microenvironment implicated in neutrophil development. To identify possible mechanisms underpinning disruption of stroma as a site of drug bioactivation, toxicity was induced in vitro. Therefore metabolite generation procedures utilizing HOCl or HRP–H₂O₂ as primary components involved in clozapine metabolism were adapted for stromal culture and coupled with viability determinations. Drug oxidation by HOCl was less toxic to stromal cells than HRP–H₂O₂ based methods. More specifically, clozapine bioactivation by HRP–H₂O₂ caused dose-dependent inhibition of stromal viability at therapeutically relevant concentrations. Differences in susceptibility of HAS303 and LP101 cells to the clozapine nitrenium ion were also evident. Stromal cell death was attributed to clozapine in the presence of a complete metabolising system comprising HRP and H₂O₂. In the absence of a complete metabolising system clozapine was not cytotoxic. For LP101 cells, drug plus HRP (minus H₂O₂) also induced toxicity. Importantly, other antipsychotic drugs including risperidone, olanzapine and haloperidol when bioactivated, were not cytotoxic, indicating system specificity for clozapine. Exogenous GSH, N-acetylcysteine,l-ascorbic acid, catalase, and sodium azide afforded protection to cells whereas S-methylGSH, GSSG, ketoprofen and proadifen did not. Thus functional data derived from the in vitro stromal system defined in these studies may enable further investigation of the mechanisms subserving stromal impairment in clozapine-induced agranulocytosis and direct attention to improved methods for its prevention.

Keywords: Abbreviations; ANOVA; analysis of variance; FCS; fetal calf serum; GSH; glutathione (reduced); GSSG; glutathione (disulfide); HBSS; Hanks balanced salt solution; H; ₂; O; ₂; hydrogen peroxide; HOCl; hypochlorous acid; HRP; horseradish peroxidase; IC; ₅₀; 50% inhibitory concentration; IMDM; Iscove's modified Dulbecco's medium; MPO; myeloperoxidase; NaHOCl; sodium hypochlorite; PBS; phosphate-buffered saline; SEM; standard error of the meanClozapine; Bioactivation; Stromal cells; Toxicity; Agranulocytosis; Schizophrenia

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Biochemical Pharmacology (v.72, #6)