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Investigating the effect of emetic compounds on chemotaxis in Dictyostelium identifies a non-sentient model for bitter and hot tastant research.

ABSTRACT: Novel chemical entities (NCEs) may be investigated for emetic liability in a range of unpleasant experiments involving retching, vomiting or conditioned taste aversion/food avoidance in sentient animals. We have used a range of compounds with known emetic /aversive properties to examine the possibility of using the social amoeba, Dictyostelium discoideum, for research into identifying and understanding emetic liability, and hence reduce adverse animal experimentation in this area. Twenty eight emetic or taste aversive compounds were employed to investigate the acute (10 min) effect of compounds on Dictyostelium cell behaviour (shape, speed and direction of movement) in a shallow chemotaxic gradient (Dunn chamber). Compound concentrations were chosen based on those previously reported to be emetic or aversive in in vivo studies and results were recorded and quantified by automated image analysis. Dictyostelium cell motility was rapidly and strongly inhibited by four structurally distinct tastants (three bitter tasting compounds--denatonium benzoate, quinine hydrochloride, phenylthiourea, and the pungent constituent of chilli peppers--capsaicin). In addition, stomach irritants (copper chloride and copper sulphate), and a phosphodiesterase IV inhibitor also rapidly blocked movement. A concentration-dependant relationship was established for five of these compounds, showing potency of inhibition as capsaicin (IC(50) = 11.9 ± 4.0 µM) > quinine hydrochloride (IC(50) = 44.3 ± 6.8 µM) > denatonium benzoate (IC(50) = 129 ± 4 µM) > phenylthiourea (IC(50) = 366 ± 5 µM) > copper sulphate (IC(50) = 1433 ± 3 µM). In contrast, 21 compounds within the cytotoxic and receptor agonist/antagonist classes did not affect cell behaviour. Further analysis of bitter and pungent compounds showed that the effect on cell behaviour was reversible and not cytotoxic, suggesting an uncharacterised molecular mechanism of action for these compounds. These results therefore demonstrate that Dictyostelium has potential as a non-sentient model in the analysis of the molecular effects of tastants, although it has limited utility in identification of emetic agents in general.

SUBMITTER: Robery S

PROVIDER: S-EPMC3169598 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Investigating the effect of emetic compounds on chemotaxis in Dictyostelium identifies a non-sentient model for bitter and hot tastant research.

Robery Steven S Mukanowa Janina J Percie du Sert Nathalie N Andrews Paul L R PL Williams Robin S B RS

PloS one 20110908 9

Novel chemical entities (NCEs) may be investigated for emetic liability in a range of unpleasant experiments involving retching, vomiting or conditioned taste aversion/food avoidance in sentient animals. We have used a range of compounds with known emetic /aversive properties to examine the possibility of using the social amoeba, Dictyostelium discoideum, for research into identifying and understanding emetic liability, and hence reduce adverse animal experimentation in this area. Twenty eight e ...[more]

PMID: 21931717

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Project description:Bronchodilators are a standard medicine for treating airway obstructive diseases, and ?2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than ?2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca(2+) signals, as revealed in cultured ASM cells, to activate large-conductance Ca(2+)-activated K(+) channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca(2+) events nor alter spontaneous local Ca(2+) transients. Interestingly, they increase global intracellular [Ca(2+)]i, although to a much lower level than bronchoconstrictors. We show that these Ca(2+) changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade (i.e., TAS2R-gustducin-phospholipase C? [PLC?]- inositol 1,4,5-triphosphate receptor [IP3R]), and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in [Ca(2+)]i induced by bronchoconstrictors, and this lowering of the [Ca(2+)]i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca(2+) channels (VDCCs), resulting in reversal in [Ca(2+)]i, and this inhibition can be prevented by pertussis toxin and G-protein ?? subunit inhibitors, but not by the blockers of PLC? and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca(2+) signaling pathways via G?? to increase [Ca(2+)]i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in [Ca(2+)]i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants.

Project description:BackgroundRas proteins are guanine-nucleotide-binding enzymes that couple cell surface receptors to intracellular signaling pathways controlling cell proliferation and differentiation, both in lower and higher eukaryotes. They act as molecular switches by cycling between active GTP and inactive GDP-bound states, through the action of two classes of regulatory proteins: a) guanine nucleotide exchange factor (GEFs) and b) GTP-ase activating proteins (GAPs). Genome wide analysis of the lower eukaryote Dictyostelium discoideum revealed a surprisingly large number of Ras Guanine Nucleotide Exchange Factors (RasGEFs). RasGEFs promote the activation of Ras proteins by catalyzing the exchange of GDP for GTP, thus conferring to RasGEFs the role of main activator of Ras proteins. Up to date only four RasGEFs, which are all non-redundant either for growth or development, have been characterized in Dictyostelium. We report here the identification and characterization of a fifth non-redundant GEF, RasGEFM.ResultsRasGEFM is a multi-domain protein containing six poly-proline stretches, a DEP, RasGEFN and RasGEF catalytic domain. The rasGEFM gene is differentially expressed during growth and development. Inactivation of the gene results in cells that form small, flat aggregates and fail to develop further. Expression of genes required for aggregation is delayed. Chemotaxis towards cAMP is impaired in the mutant, due to inability to inhibit lateral pseudopods. Endogenous cAMP accumulates during early development to a much lower extent than in wild type cells. Adenylyl cyclase activation in response to cAMP pulses is strongly reduced, by contrast guanylyl cyclase is stimulated to higher levels than in the wild type. The actin polymerization response to cAMP is also altered in the mutant. Cyclic AMP pulsing for several hours partially rescues the mutant. In vitro experiments suggest that RasGEFM acts downstream of the cAMP receptor but upstream of the G protein.ConclusionThe data indicate that RasGEFM is involved in the establishment of the cAMP relay system. We propose that RasGEFM is a component of a Ras regulated pathway, which integrate signals acting as positive regulator for adenylyl cyclase and negative regulator for guanylyl cyclase. Altered guanylyl cyclase, combined with defective regulation of actin polymerization, results in altered chemotaxis.

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Investigating the effect of emetic compounds on chemotaxis in Dictyostelium identifies a non-sentient model for bitter and hot tastant research.

Publications

Investigating the effect of emetic compounds on chemotaxis in Dictyostelium identifies a non-sentient model for bitter and hot tastant research.

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