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 μM), bisindolylmaleimide (1 μM) or chelerythrine (1 μM), irreversibly suppressed thrombin-induced [Ca²⁺]_i oscillations. Thereafter application of 2,5-di(tert-butyl)-1,4-benzohydroquinone (t-BuBHQ, 20 μM) or thapsigargin (1 μM) (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-BuBHQ 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. Received: 5 July 1996 / Received after revision and accepted: 7 October 1996