Cloning and characterization of genes encoding alpha and beta subunits of glutamate-gated chloride channel protein in Cylicocyclus nassatus.

The invertebrate glutamate-gated chloride channels (GluCls) are receptor molecules and targets for the avermectin-milbemycin (AM) group of anthelmintics. Mutations in GluCls are associated with ivermectin resistance in the soil dwelling nematode Caenorhabditis elegans and the parasitic nematode Cooperia oncophora. In this study, full-length cDNAs encoding alpha and beta subunits of GluCl were cloned and sequenced in Cylicocyclus nassatus, a common and important cyathostomin nematode parasite of horses. Both genes possess the sequence characteristics typical of GluCls, and phylogenetic analysis confirms that these genes are evolutionarily closely related to GluCls of other nematodes and flies. Complete coding sequences of C. nassatus GluCl-alpha and GluCl-beta were subcloned into pTL1 mammalian expression vector, and proteins were expressed in COS-7 cells. Ivermectin-binding characteristics were determined by incubating COS-7 cell membranes expressing C. nassatus GluCl-alpha and GluCl-beta proteins with [(3)H]ivermectin. In competitive binding experiments, fitting the data to a one site competition model, C. nassatus GluCl-alpha was found to bind [(3)H]ivermectin with a high amount of displaceable binding (IC(50)=208 pM). Compared to the mock-transfected COS-7 cells, the means of [(3)H]ivermectin binding were significantly different for C. nassatus GluCl-alpha and the Haemonchus contortus GluCl (HcGluCla) (p=0.018 and 0.023, respectively) but not for C. nassatus GluCl-beta (p=0.370). This is the first report of orthologs of GluCl genes and in vitro expression of an ivermectin-binding protein in a cyathostomin species. These data suggest the likelihood of a similar mechanism of action of AM drugs in these parasites, and suggest that mechanisms of resistance may also be similar.

On the use of neuro-2a neuroblastoma cells versus intact neurons in primary culture for neurotoxicity studies.

Neuroblastoma cell lines have been used extensively to screen novel compounds for neurotoxic properties and associated mechanisms. Such transformed cell lines often display morphological, developmental, and signaling characteristics that are substantially different from the parental cell type. Consequently, the response of neuroblastoma cells to toxin exposure may differ from that of neurons. An appreciation of the pharmacological and functional differences between neurons and neuron-like cell lines is therefore essential when interpreting data derived from neuroblastoma-based assays. We have compared the effects of several neurotoxins on Ca2+ homeostasis and cell viability in cerebellar granule neurons (CGN) and a neuroblastoma cell line (Neuro-2a). To explore the mechanisms underlying differential sensitivity of intact neurons and neuroblastoma cells to neurotoxins, we also compared CGN and Neuro-2a cells for expression of voltage-gated sodium channels (VGSC) and N-methyl-D-aspartate receptors (NMDAR). Cytotoxic potency in neurons was several orders of magnitude greater for Caribbean-ciguatoxin-1 (C-CTX-1) than either domoate (Dom) or brevetoxin-2 (PbTx-2). In addition, the cytotoxic potency of C-CTX-1 was two orders of magnitude greater in CGN than in Neuro-2a cells. The effect of C-CTX-1 and Dom on calcium homeostasis was compared in fluo-3 loaded neurons. Dom caused an elevation in intracellular calcium ([Ca2+]i) at concentrations that paralleled the concentration/response relationship for cytotoxicity in CGN. Conversely, C-CTX-1 did not elevate [Ca2+]i within the dynamic concentration range for cell death. The discordance of the concentration/response relationships for C-CTX-1 induced cytotoxicity and [Ca2+]i elevation suggests that acute C-CTX-1 cytotoxicity may involve mechanisms other than Ca2+ load. C-CTX-1-induced elevation of [Ca2+]i in neurons was dependent on activation of NMDAR and the reverse mode of operation of the Na+/Ca2+ exchanger. These data demonstrate that, although C-CTX-1, domoate, and PbTx-2 share the ability to produce neurotoxicity and mobilize calcium, their respective molecular targets and mechanisms of neurotoxicity differ. Neuro-2a cells that were not pretreated with veratridine and ouabain were insensitive to C-CTX-1 and glutamatergic agonists. VGSC expression was 20-fold lower in Neuro-2a cells than in CGN, whereas NMDARs were not expressed in these neuroblastoma cells. It is therefore likely that the enhanced sensitivity of CGN, relative to Neuro-2a cells, to neurotoxins is a consequence of pronounced differences in VGSC and NMDAR expression. These results underscore the need to exercise caution in interpreting negative cytotoxicity data derived from the use of neuroblastoma cell lines.

Domoic acid neurotoxicity in cultured cerebellar granule neurons is controlled preferentially by the NMDA receptor Ca(2+) influx pathway.

We have monitored real-time alterations in [Ca(2+)](i) in fluo-3-loaded cerebellar granule neurons exposed to domoate, and ascertained the influence of pharmacological blockers of various Ca(2+) entry pathways on intracellular Ca(2+) accumulation, excitatory amino acid (EAA) release and neuronal death. Domoate produced a rapid and concentration-dependent increase in [Ca(2+)](i), the magnitude of which correlated closely with the severity of neuron loss. The increase in [Ca(2+)](i) was derived from activation of NMDA receptors, L-type voltage-sensitive calcium channels (VSCC) and the reversed mode of operation of the Na(+)/Ca(2+) exchanger. When the level of neuroprotection conferred by pharmacological manipulation of these calcium entry pathways was regressed with the corresponding reductions in [Ca(2+)](i) load, it was observed that neuronal vulnerability is controlled preferentially by NMDA receptors. This observation is consistent with our previous study of brevetoxin-induced autocrine excitotoxicity and with the source specificity hypothesis of others [J. Neurochem. 71 (1998) 2349], which suggests that elevation of [Ca(2+)](i) in the vicinity of the NMDA receptor ion channel activates processes leading to neuronal death.

Investigation of cancer cell lines for peptide receptor-targeted drug development.

Many tumors highly express specific populations of G-protein-coupled receptors (GPCRs) that could be utilized for receptor-targeted therapy. We confirmed significant quantities of mRNAs specific for certain somatostatin (SST), vasoactive intestinal peptide (VIP), and bombesin (BN) receptors in various commercially available tumor cell lines. Very few of the tumor cell lines examined displayed the high receptor-binding affinity despite exhibiting the expression of appropriate mRNAs and proteins of the cognate receptors. However, binding assays establish that some tumor cell lines, such as pancreatic cancer CFPAC-1, prostate cancer DU-145, and pancreatic carcinoid BON, demonstrate high BN receptor binding. BON cells also demonstrate high somatostatin receptor (SSTR) affinity binding. We also found that tumor cell lines, such as BON and host cells expressing SST receptor subtypes 1 or 2 (CHO-R1 or CHO-R2), underwent a decrease in cell surface receptor density in multiple passages. BON and CHO-R2 cells also rapidly internalize a significant proportion of cell surface ligand-receptor complexes. The tumor cells CFPAC-1, DU-145, and BON with high receptor binding could be useful for peptide drug studies. BON cells were further applied to test SST/BN analogs and cytotoxic conjugates. Furthermore, the in vivo antitumor assay showed that the cytotoxic conjugate CPT-SST targeting all SSTR subtypes displayed a potent tumor-suppressive ability to BON tumors expressing multiple SSTR subtypes.

Application of human pancreatic carcinoid BON cells for receptor-targeted drug development.

In our previous study, we found that several tumor cell lines displayed high receptor-specific binding affinity, one of which, the human pancreatic carcinoid BON cell line, demonstrates high affinity binding of the bombesin (BN) and somatostatin (SST) receptor-specific ligands. In the present study, BON cells, as a representative model, were further applied to evaluate various peptide analogs and cytotoxic receptor-targeted peptide conjugates. We observed quick ligand-receptor internalization in BON cells as well as high binding affinity. Furthermore, BON cells have high expression of multidrug resistance-associated genes (MDR1) and show camptothecin (CPT) resistance. Various receptor-specific cytotoxic conjugates were synthesized and evaluated in the BON cell model via in vitro and in vivo studies. We found that all the tested conjugates displayed potent antitumor ability in xenografts. Especially, the CPT conjugates, CPT-SST, and CPT-BN, are most likely to increase sensitivity to CPT-resistant BON cells. Our findings suggest that appropriately defined tumor cell lines may provide physiologically relevant cell-based evaluations of novel peptide analogs and receptor-targeted chemotherapeutics.

Involvement of caspase activation in azaspiracid-induced neurotoxicity in neocortical neurons.

Azaspiracids (AZAs) are a novel group of marine phycotoxins that have been associated with severe human intoxication. We found that AZA-1 exposure increased lactate dehydrogense (LDH) efflux in murine neocortical neurons. AZA-1 also produced nuclear condensation and stimulated caspase-3 activity with an half maximal effective concentration (EC(50)) value of 25.8 nM. These data indicate that AZA-1 triggers neuronal death in neocortical neurons by both necrotic and apoptotic mechanisms. An evaluation of the structure-activity relationships of AZA analogs on LDH efflux and caspase-3 activation demonstrated that the full structure of AZAs was required to produce necrotic or apoptotic cell death. The similar potencies of AZA-1 to stimulate LDH efflux and caspase-3 activation and the parallel structure-activity relationships of azaspiracid analogs in the two assays are consistent with a common molecular target for both responses. To explore the molecular mechanism for AZA-1-induced neurotoxicity, we assessed the influence of AZA-1 on Ca(2+) homeostasis. AZA-1 suppressed spontaneous Ca(2+) oscillations (EC(50) = 445 nM) in neocortical neurons. A distinct structure-activity profile was found for inhibition of Ca(2+) oscillations where both the full structure as well as analogs containing only the FGHI domain attached to a phenyl glycine methyl ester moiety were potent inhibitors. The molecular targets for inhibition of spontaneous Ca(2+) oscillations and neurotoxicity may therefore differ. The caspase protease inhibitor Z-VAD-FMK produced a complete elimination of AZA-1-induced LDH efflux and nuclear condensation in neocortical neurons. Although the molecular target for AZA-induced neurotoxicity remains to be established, these results demonstrate that the observed neurotoxicity is dependent on a caspase signaling pathway.

Neurotoxic meroditerpenoids from the tropical marine brown alga Stypopodium flabelliforme.

Brine shrimp toxicity and TLC analysis guided the isolation of five new and biologically active meroditerpenoids [2beta,3alpha-epitaondiol (1), flabellinol (2), flabellinone (3), stypotriolaldehyde (4), and stypohydroperoxide (5)] along with five known compounds from the marine brown alga Stypopodium flabelliforme collected in Papua New Guinea. The planar structures of compounds 1-5 were determined by extensive spectroscopic analysis (1D and 2D NMR, LRMS, HRMS, IR, and UV), while relative configuration was determined by 1D and 2D NOE experiments. X-ray crystallography confirmed the relative configuration of 2beta,3alpha-epitaondiol (1), and the modified Mosher's ester method was used to establish its absolute configuration. All of the new metabolites were moderately toxic to murine neuro-2a cells (LC50 2-25 microM), and three [2beta,3alpha-epitaondiol (1), flabellinol (2), and flabellinone (3)] possessed potent sodium channel blocking activity. Stypotriolaldehyde (4) had a biphasic effect on the concentration of intracellular Ca2+ in rat cerebellar granule neurons (CGN). The previously known compound, stypoldione (6), also modulated intracellular calcium concentration and was cytotoxic in CGN. Metabolites 2beta,3alpha-epitaondiol (1), flabellinol (2), and flabellinone (3) displayed moderate cytotoxicity to the NCI-H460 human lung cancer cell line.