8 - Diethylamino - octyl - 3, 4, 5 - trimethoxyben-zoate (TMB-8) is known to inhibit mobilization of calcium from intracellular stores but, more recently, other cellular effects have been described. In the present study, the effects of TMB-8 on cytosolic free calcium [Ca2+]ilevels in FRTL-5 rat thyroid cells were determined using the fluorescent dye, Indo-1. TMB-8 increased [Ca2+]iin a dose-dependent manner, with a maximum rise from 120 ± 7 nM to 229 ± 16 nM (90 ± 5% increase) at 5 x 10~4m. This effect was considerably reduced in Ca2+free buffer, demonstrating a dependency upon extracellular calcium influx but not upon membrane potential and which did not involve the Na+/Ca2+exchanger. In Ca2+free buffer TMB-8, at a dose which did not affect [Ca2+]i, completely prevented norepinephrine (10-5M) from mobilizing intracellular Ca2+. To determine whether TMB-8 affected differentiated functions, iodide uptake and efflux studies were performed with125I. TMB-8 (10-4) inhibited iodide uptake by approximately 40% without affecting efflux. At 10-3M TMB-8, efflux was also enhanced. These studies demonstrate that TMB-8 has at least two effects on [Ca2+]i, promoting calcium influx and preventing a-l adrenergic mobilization from intracellular stores. TMB-8 also has multiple effects on125I transport, both inhibiting influx and increasing efflux. The results emphasize the importance of characterizing the behavior of this compound in any cell system before using it as a biological probe.
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