Redistribution of Ca²⁺among cytosol and organella during stimulation of bovine chromaffin cells

Abstract

Recent results indicate that Ca2+ transport by organella contributes to shaping Ca2+ signals and exocytosis in adrenal chromaffin cells. Therefore, accurate measurements of [Ca2+] inside cytoplasmic organella are essential for a comprehensive analysis of the Ca2+ redistribution that follows cell stimulation. Here we have studied changes in Ca2+ inside the endoplasmic reticulum, mitochondria, and nucleus by imaging aequorins targeted to these compartments in cells stimulated by brief depolarizing pulses with high K+ solutions. We find that Ca2+ entry through voltage-gated Ca2+ channels generates subplasmalemmal high [Ca2+]c domains adequate for triggering exocytosis. A smaller increase of [Ca2+]c is produced in the cell core, which is adequate for recruitment of the reserve pool of secretory vesicles to the plasma membrane. Most of the Ca2+ load is taken up by a mitochondrial pool, M1, closer to the plasma membrane; the increase of [Ca2+]M stimulates respiration in these mitochondria, providing local support for the exocytotic process. Relaxation of the [Ca2+]c transient is due to Ca2+ extrusion through the plasma membrane. At this stage, mitochondria release Ca2+ to the cytosol through the Na+/Ca2+ exchanger, thus maintaining [Ca2+]c discretely increased, especially at core regions of the cell, for periods that outlast the duration of the stimulus.

Mitochondria and chromaffin cell function

“…When we measured the rate of [Ca 2+ ] C increase in chromaffin cells depolarised with high K + , we found values of 1 μmol · l cells −1 · s −1 [55]. This is only 1 of 700 of the estimated Ca 2+ entry.…”

“…The difference was due to pumping out Ca 2+ : (1) to the extracellular medium through the plasma membrane (20-100 μmol · l cells −1 · s −1 ); (2) inside ER (70-80 μmol · l cells −1 · s −1 ); and (3) inside mitochondria, which is, by far, the largest component, more than 90% of the total entry ( Fig. 1) [55].…”

“…Taking into account the binding to cytosolic Ca 2+ buffers, we would expect a rate of 700/400 17.5 μmol · l cells −1 · s −1 , which is still much larger than the measured value, suggesting that more than 90% of the entering Ca 2+ is muffled [54] by transport out of the cytosol. Using aequorins targeted to different organelles delivered by a herpes virus [2], we were able to measure the contribution of the different transport processes [55]. Results are summarized in Fig.…”

“…Uptake by SERCA increases exponentially with [Ca 2+ ] C and can reach maximal rates 70-80 μmol · l cells [3,55,60]. At the steady state, the ER fills to a level of 500-1,000 μM [Ca 2+ ].…”

“…On the other hand, organelles interrupt physically the diffusion pathways and make them much longer and tortuous. In addition, many organelles avidly take up Ca 2+ and drain the Ca 2+ wave; on the contrary, other organelles amplify the [Ca 2+ ] C signal by releasing Ca 2+ [3,5,7,55]. In this way high [Ca 2+ ] C microdomains (HCMD) are built at the secretion site whereas [Ca 2+ ] C near the resting level is preserved at the cell core.…”