STAUROSPORINE INDUCED CA-2+ INCREASE IN DDT1MF-2 SMOOTH-MUSCLE CELLS

The free calcium concentrations in nucleus ([Ca2+]n) and cytoplasm ([Ca2+]c) of cultured DDT1MF-2 smooth muscle cells were estimated using the fluorescent dye indo-1 and laser confocal microscopy. The alkaloid staurosporine mainly increased [Ca2+]c during the initial minutes of stimulation and the nucleocytoplasmic gradient was maintained. Thereafter [Ca2+]n increased further while [Ca2+]c decreased, resulting after 10 min in a reversion of the nucleocytoplasmic gradient. Staurosporine increased the Ca2+ influx but also released intracellular Ca2+. In Ca2+-free solution no inversion of the nucleocytoplasmic gradient was seen in most cells. Ca2+ increased more rapidly in the area of the perinuclear sarcoplasmic reticulum (SR) than in the central cytoplasm. This is suggestive of the release of Ca2+ from this region. Ca2+ concentration remained also enhanced near the plasmalemma during this time period. This might indicate that the efflux mechanism is also affected. It is unlikely that these changes are due to inhibition of protein kinase C, since phorbol esters are ineffective and since downregulation by prolonged incubation with phorbol 12-myristate 13-acetate only affected the time periods required to attain the described [Ca2+]c and [Ca2+]n peaks. The slow time course of the staurosporine effects allowed demonstration of the spatiotemporal Ca2+ gradients between the nucleus and the cytoplasm and also inside the cytosol. The different time course of the increases in [Ca2+]c and [Ca2+]n suggests that two different Ca2+ mobilization events are present. The occurrence of an inversion of the nucleocytoplasmic gradient whereby [Ca2+]n increased while [Ca2+]c values decreased indicates that the increase in [Ca2+]n might result partially from release sites inside the nucleus. Because in Ca2+-free conditions only part of the cell population presented this inversion, further investigations are required to establish the contribution of extracellular Ca2+ to these events.