Activity of protein kinase C is necessary for sustained thrombin-induced [Ca2+](i) oscillations in rat glioma cells

The aim of the present study was to examine the possible role of protein kinase C (PKC) in thrombin-induced Ca2+ signalling. As shown before, continuous superfusion of rat glioma cells with thrombin caused sustained [Ca2+](i) oscillations through activation of cell surface receptors [Czubayko U, Reiser G (1995) Neuroreport 6: 1249]. These oscillations were inhibited by protease nexin-1. Addition of PKC inhibitors, i. e. staurosporine (0.2-20 mu M), bisindolylmaleimide (1 mu M) or chelerythrine (1 mu M), irreversibly suppressed thrombin-induced [Ca2+](i) oscillations. Thereafter application of 2,5-di(tert-butyl)-1 ,4-benzohydroquinone (t-BuBHQ, 20 mu M) or thapsigargin (1 mu M) (inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase) caused no [Ca2+](i) response, indicating that intracellular Ca2+ stores were completely empty. We tested whether PKC affects the refilling of internal Ca2+ stores in thrombin-stimulated cells, by monitoring the amount of Ca2+ release caused by t-Bu-BHQ in the presence or absence of PKC inhibitors or activators. The amount of Ca2+ released by t-BuBHQ, which was normalized by comparison with the thrombin-induced Ca2+ response, was decreased by simultaneous incubation with staurosporine or chelerythrine, but enhanced with the PKC activator oleoyl acetyl glycerol. Furthermore, the capacitative Ca2+ entry was reduced by inhibition or downregulation, and increased by activation, of PKC. Capacitative Ca2+ entry was induced in these experiments by depletion of Ca2+ stores by the addition of thapsigargin or t-BuBHQ. In contrast, the inhibition of PKC during thrombin-induced depletion of intracellular stores did not influence the Ca2+ entry but nearly completely abolished the refilling of the internal stores. Thus we conclude that during thrombin receptor stimulation activation of PKC is required to maintain the refilling of intracellular Ca2+ stores for sustained [Ca2+](i) oscillations. Thus, the control by PKC of the capacitative Ca2+ entry is apparently different depending on whether it is induced by sarco/endoplasmic reticulum Ca2+-ATPase inhibition or by activation of the thrombin receptor.