Development and dissipation of Ca(2+) gradients in adrenal chromaffin cells.
We used pulsed laser imaging to measure the development and dissipation of Ca(2+) gradients evoked by the activation of voltage-sensitive Ca(2+) channels in adrenal chromaffin cells. Ca(2+) gradients appeared rapidly (<5 ms) upon membrane depolarization and dissipated over several hundred milliseconds after membrane repolarization. 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 Ca(2+) uptake by the endoplasmic reticulum (thapsigargin) and mitochondria (carbonylcyanide p-trifluoro-methoxyphenylhydrazone/oligomycin) had no effect on the size of Ca(2+) changes or the rate of gradient dissipation, suggesting that passive endogenous Ca(2+) buffers are responsible for the slow Ca(2+) redistribution. We used a radial diffusion model incorporating Ca(2+) diffusion and binding to intracellular Ca(2+) buffers to simulate Ca(2+) 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 Ca(2+) buffer, with a buffer capacity on the order of 1000 and appropriate affinity and kinetics, approximated the size of the Ca(2+) increases and rate of dissipation of the measured gradients. Finally, simulations without exogenous buffer suggest that the Ca(2+) signal due to Ca(2+) channel activation is restricted by the endogenous buffer to a space less than 1 microm from the cell membrane.
Marengo
F D
FD
Department of Physiology, UCLA School of Medicine, Los Angeles, California 90095, USA.
Monck
J R
JR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002021:Buffers; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling; D002417:Cattle; D019439:Chromaffin Cells / Q000378:metabolism*; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D008564:Membrane Potentials; D008954:Models, Biological
D002021:Buffers; D015220:Calcium Channels; D000255:Adenosine Triphosphate; D002118:Calcium