Abstract
Exocytosis in adrenal chromaffin cells is strongly influenced by the pattern of stimulation. To understand the dynamic and spatial properties of the underlying Ca(2+) signal, we used pulsed laser Ca(2+) imaging to capture Ca(2+) gradients during stimulation by single and repetitive depolarizing stimuli. Short single pulses (10-100 ms) lead to the development of submembrane Ca(2+) gradients, as previously described (F. D. Marengo and J. R. Monck, 2000, Biophysical Journal, 79:1800-1820). Repetitive stimulation with trains of multiple pulses (50 ms each, 2Hz) produce a pattern of intracellular Ca(2+) increase that progressively changes from the typical Ca(2+) gradient seen after a single pulse to a Ca(2+) increase throughout the cell that peaks at values 3-4 times higher than the maximum values obtained at the end of single pulses. After seven or more pulses, the fluorescence increase was typically larger in the interior of the cell than in the submembrane region. The pattern of Ca(2+) gradient was not modified by inhibitors of Ca(2+)-induced Ca(2+) release (ryanodine), inhibitors of IP(3)-induced Ca(2+) release (xestospongin), or treatments designed to deplete intracellular Ca(2+) stores (thapsigargin). However, we found that the large fluorescence increase in the cell interior spatially colocalized with the nucleus. These results can be simulated using mathematical models of Ca(2+) redistribution in which the nucleus takes up Ca(2+) by active or passive transport mechanisms. These results show that chromaffin cells can respond to depolarizing stimuli with different dynamic Ca(2+) signals in the submembrane space, the cytosol, and the nucleus.