Development and dissipation of Ca2+ gradients in adrenal chromaffin cells

Abstract

We used pulsed laser imaging to measure the development and dissipation of Ca2+ gradients evoked by the activation of voltage-sensitive Ca2+ channels in adrenal chromaffin cells. Ca2+ gradients appeared rapidly (<5 ms) upon membrane depolarization and dissipated over several hundred milliseconds after membrane repollarization. Dissipation occurred with an initial fast phase, as the steep gradient near the membrane collapsed, and a slower phase as the remaining shallow gradient dispersed. Inhibition of active Ca2+ uptake by the endoplasmic reticulum (thapsigargin) and mitochondria (carbonylcyanide p-trifluoro-methoxyphenylhydrazone/oligomycin) had no effect on the size of Ca2+ changes or the rate of gradient dissipation, suggesting that passive endogenous Ca2+ buffers are responsible for the slow Ca2+ redistribution. We used a radial diffusion model incorporating Ca2+ diffusion and binding to intracellular Ca2+ buffers to simulate Ca2+ gradients. We included a 3D optical sectioning model, simulating the effects of out-of-focus light, to allow comparison with the measured gradients. Introduction of a high-capacity immobile Ca2+ buffer, with a buffer capacity on the order of 1000 and appropriate affinity and kinetics, approximated the size of the Ca2+ increases and rate of dissipation of the measured gradients. Finally, simulations without exogenous buffer suggest that the Ca2+ signal due to Ca2+ channel activation is restricted by the endogenous buffer to a space less than 1 μm from the cell membrane.