Tri‐n‐butyltin Increases Intracellular Ca²⁺ in Mouse Thymocytes: A Flow‐Cytometric Study Using Fluorescent Dyes for Membrane Potential and Intracellular Ca²⁺

Abstract

Effects of tri-n-butyltin (TBT) on mouse thymocytes were examined using a flow-cytometer and fluorescent dyes for membrane potential and intracellular Ca2+ ([Ca2+]i). TBT at concentrations from 1 x 10(-7) M to 3 x 10(-7) M caused hyperpolarization in thymocytes during 30 min. after drug application in a time- and dose-dependent manner. Further increase in TBT concentration (to 1 x 10(-6) M) made hyperpolarization of thymocytes more profound within 5 min. after application, thereafter gradually depolarized them during the next 25 min. TBT at 3 x 10(-8) M or more (up to 1 x 10(-6) M) increased the [Ca2+]i of thymocytes. After reaching maximum [Ca2+]i at the various TBT concentrations used within 5 min. after drug application, the [Ca2+]i slightly decreased in a time-dependent manner. Effects of TBT on membrane potential and the [Ca2+]i were greatly reduced under nominal external Ca(2+)-free condition. Results suggest that TBT can promote Ca(2+)-influx to thymocytes, resulting in hyperpolarization by activation of Ca(2+)-dependent K+ current. The increase in [Ca2+]i by TBT may be related to its cytotoxic action on thymocytes.

Some characteristics of membrane Cd2+ transport in rat thymocytes: an analysis using Fluo-3

“…The technique to dissociate thymocytes from murine thymic glands was similar to that previously described (Chikahisa and Oyama 1992;Chikahisa et al 1996). Thymus glands dissected from 4-5 week old Wistar rats were sliced at a thickness of 400-500 lm with a razor.…”

“…To monitor the change in membrane potential of thymocytes, bis-(1,3-dibutyl barbituric acid)trimethine oxonol (Oxonol, Molecular Probes Inc.) was used (Chikahisa and Oyama 1992). This probe was initially dissolved in DMSO and the DMSO solution was added into the cell suspension to achieve a final Oxonol concentration of 300 nM.…”

Cytotoxic actions of N-(2,4,6-trichlorophenyl)maleimide (IT-354), an antifouling agent, in rat thymic lymphocytes

“…2B. Trin-butyltin, the most toxic antifoulant, increased intracellular concentrations of Ca 2+ and Zn 2+ (Chikahisa and Oyama, 1992;Oyama et al, 2009). The effect of IT-354 on FluoZin-3 fluorescence, an indicator of intracellular Zn 2+ , was also examined.…”

Elevation of Intracellular Ca2+ Level by Triclosan in Rat Thymic Lymphocytes: Increase in Membrane Ca2+ Permeability and Induction of Intracellular Ca2+ Release

“…Cell suspension was prepared similar to that previously reported. 23,24) In brief, thymus glands dissected from ether-anesthetized rats were sliced to 400-500 µm thickness with a razor under cold conditions (3-4 • C). The slices were triturated by gently shaking them in chilled Ca 2+ -free Tyrode's solution (150 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 5 mM glucose, 5 mM HEPES adjusted to a pH of 7.3-7.4) to dissociate thymocytes.…”

“…Fluorescence Measurements of Cellular and Membrane Parameters --The methods for measurements of cellular and membrane parameters by using a flow cytometer equipped with an argon laser (CytoACE-150, JASCO, Tokyo, Japan) and fluorescent probes were similar to those previously described. 23,24) The fluorescence was analyzed using the JASCO software (Ver. 3.06).…”