The nuclear envelope of resting C6 glioma cells is able to release and uptake Ca²⁺ in the absence of chemical stimulation

Abstract

 Many agonists evoke events in the cell nucleus through the control of Ca²⁺ signals. Recent studies using isolated nuclei have indicated that the nuclear envelope is a store for nuclear Ca²⁺. However, the release of Ca²⁺ directly from the nuclear envelope in living cells has never been reported. In the present study, we have investigated the changes of Ca²⁺ signals at the cyto-nucleoplasmic interface of rat C6 glioma cells using confocal microscopy. Digital imaging indicates that fluo-3, a Ca²⁺-sensitive fluorescent probe, was concentrated in or around the nuclear envelope. Our experiments also revealed that C6 cells at rest produced spontaneous Ca²⁺ spikes in the absence of chemical stimulation. The amplitude of the repetitive Ca²⁺ spikes was higher at the nuclear envelope than in the whole cell or cytosol. After image subtraction, circular rims of Ca²⁺ release and uptake were seen at the outer boundary of the nucleus. When the cells were treated with thapsigargin (2 μM), a specific Ca²⁺-ATPase inhibitor, a long-lasting Ca²⁺ release was observed at the nuclear envelope. Moreover, most of the released Ca²⁺ was directed inwardly to the nucleoplasm with little outward diffusion. Our results thus indicate: (1) that the nuclear envelope is a Ca²⁺ store that possesses the ability to discharge and sequestrate Ca²⁺; and (2) the Ca²⁺-releasing channels are present in the inner nuclear membrane.