Cytotoxic action of tri-n-butyltin on dissociated rat cerebellar neurones: a flow-cytometric study.

The effects of tri-n-butyltin on cell viability and intracellular Ca2+ concentration ([CA2+]i) were examined by a flow cytometer in rat cerebellar neurones to reveal the contribution of tri-n-butyltin-induced increased in the [Ca2+]i to its cytotoxicity. Tri-n-Butyltin decreased the cell viability in association with increased [Ca2+]i at concentrations of 0.3 microM or more. Decrease or increase of the extracellular Ca2+ concentration ([Ca2+]o) respectively decreased or increased the cell viability. However, cell viability in the presence of ionomycin which increased [Ca2+]i more significantly than tri-n-butyltin was higher than that in the presence of tri-n-butyltin. Tri-n-butyltin also decreased cell viability under nominally [Ca2+]o-free conditions although the [Ca2+]i increase by tri-n-butyltin was still lower than the control [Ca2+]i under normal [Ca2+]o (2 mM). Therefore, it is unlikely that neuronal death induced by tri-n-butyltin is entirely dependent on the tri-n-butyltin-induced increase in [Ca2+]i.

Methylmercury induces Ca(2+)-dependent hyperpolarization of mouse thymocytes: a flow cytometric study using fluorescent dyes.

The effect of methylmercury on mouse thymocytes was examined using fluorescent dyes for membrane potential and intracellular Ca2+. Methylmercury at concentrations of 1 microM or higher (up to 30 microM) produced hyperpolarization in a dose-dependent fashion. Charybdotoxin and quinine, but not 4-aminopyridine and tetraethylammonium, greatly suppressed methylmercury-induced hyperpolarization. Removal of external Ca2+ reduced the degree of hyperpolarization. Pretreatment of thymocytes with A23187 under Ca(2+)-free conditions abolished the hyperpolarization induced by methylmercury. Under both normal and Ca(2+)-free conditions methylmercury increased the intracellular concentration of Ca2+. The results suggest that the increase in intracellular Ca2+ is mediated through a Ca2+ release from intracellular stores as well as through influx of external Ca2+. Therefore, it is likely that methylmercury increases the intracellular concentration of Ca2+, resulting in activation of Ca(2+)-dependent K+ conductance of mouse thymocytes.

Methylmercury-induced augmentation of oxidative metabolism in cerebellar neurons dissociated from the rats: its dependence on intracellular Ca2+.

Effect of methylmercury chloride on oxidative metabolism of cerebellar neurons dissociated from the rats was examined using 2',7'-dichlorofluorescin (DCFH) which is oxidized by cellular hydrogen peroxide to be a fluorescent compound (DCF) and fluo-3, an indicator for intracellular Ca2+ concentration ([Ca2+]i). Methylmercury at 1 microM or less did not affect DCF fluorescence of cerebellar neurons. Further increase in concentration of methylmercury (up to 30 microM) induced changes in DCF fluorescence. Thus, DCF fluorescence was slightly attenuated during 5 min after applying methylmercury to the neurons, indicating a decrease in oxidation of DCFH. Thereafter, DCF fluorescence was time-dependently augmented in continued presence of methylmercury, indicating an increase in DCFH oxidation. Although methylmercury-induced augmentation of DCF fluorescence was greatly suppressed under external Ca(2+)-free condition, it was not the case for methylmercury-induced attenuation of DCF fluorescence. Methylmercury at 3 microM or more dose-dependently increased the [Ca2+]i. Results suggest that methylmercury increases intracellular Ca2+ in cerebellar neurons, resulting in an increase in formation of reactive oxygen species that may contribute to cell injury.

Isolation and characterization of a neutrophil chemotactic factor from Tritrichomonas foetus organisms.

A neutrophil chemotactic factor (TfNCF) was isolated from the crude extract of Tritrichomonas foetus organisms by a combination of anion-exchange chromatography on DE52 and gel filtration on Sephacryl S200. TfNCF showed homogenicity by both PAGE and SDS-PAGE. The molecular weight of TfNCF was estimated to be 22 and 24 kDa, by Sephacryl S200 gel chromatography and by SDS-PAGE under reducing conditions. Immunization of TfNCF caused almost complete protection against T. foetus infection in mice. Western blot analysis probed with anti-TfNCF antibody showed that the epitopes on TfNCF were not commonly shared on the other components of T. foetus organisms nor other helminthous parasite-derived components. Furthermore, pre-incubation of neutrophils with antigens of other helminthous parasites or N-formylmethionyl-leucyl-phenylalanine did not affect the neutrophil chemotactic activity for TfNCF. These results suggest that TfNCF is a novel NCF consisting of unique epitopes for both antigenicity and neutrophil chemotactic activity. The role of NCF in the initiation of the immune response is discussed.

Characterization of the triphenyltin-induced increase in intracellular Ca2+ of mouse thymocytes: comparison with the action of A23187.

The properties of triphenyltin (TPT) in increasing intracellular Ca2+ ([Ca2+]i) of thymocytes was studied, in comparison with those of A23187, by the use of fluorescent dyes to monitor membrane potential and [Ca2+]i. Both 1 microM TPT and 30 nM A23187 increased the [Ca2+]i associated with the hyperpolarization mediated by Ca(2+)-dependent K+ conductance. The time course for the TPT-induced increase in the [Ca2+]i was much slower than that of A23187. When the external Ca2+ ([Ca2+]o) was removed, TPT produced a slight, but persistent, increase in the [Ca2+]i while A23187 caused only a transient increase in the [Ca2+]i. Reintroduction of Ca2+ to the external solution produced an increase in [Ca2+]i in both cases. Therefore, these results suggested that the increase in the [Ca2+]i of thymocytes induced by TPT and A23187 was dependent on the presence of [Ca2+]o and an intracellular Ca store. The potency of TPT in increasing the [Ca2+]i was greater than those of diphenyltin and monophenyltin, suggesting an involvement of the lipophilic property of organotins in increasing [Ca2+]i. The TPT-induced increase in the [Ca2+]i may be partly responsible for the toxicity of TPT on organs and/or organ systems.

Cytotoxic action of triphenyltin on mouse thymocytes: a flow-cytometric study using fluorescent dyes for membrane potential and intracellular Ca2+.

Effects of triphenyltin on mouse thymocytes were examined using fluorescent dyes to monitor membrane potential and intracellular Ca2+. Triphenyltin at 3 x 10(-7) M to 1 x 10(-6) M hyperpolarized thymocytes and depolarized them at 3 x 10(-6) M or more, associated with increasing intracellular Ca2+. Hyperpolarization was suppressed by quinine, but not by tetraethylammonium and 4-aminopyridine, suggesting the involvement of Ca(2+)-activated K+ current. Triphentyltin failed to hyperpolarize thymocytes in Ca(2+)-free solution. Results indicate that triphenyltin promotes Ca(2+)-influx to thymocytes. Such an action of triphenyltin may be related to the immunotoxicity of organotins.