Novel features on the regulation by mitochondria of calcium and secretion transients in chromaffin cells challenged with acetylcholine at 37°C

Abstract

From experiments performed at room temperature, we know that the buffering of Ca2+ by mitochondria contributes to the shaping of the bulk cytosolic calcium transient ([Ca2+]c) and secretion transients of chromaffin cells stimulated with depolarizing pulses. We also know that the mitochondrial Ca2+ transporters and the release of catecholamine are faster at 37°C with respect to room temperature. Therefore, we planned this investigation to gain further insight into the contribution of mitochondrial Ca2+ buffering to the shaping of [Ca2+]c and catecholamine release transients, using some novel experimental conditions that have not been yet explored namely: (1) perifusion of bovine chromaffin cells (BCCs) with saline at 37°C and their repeated challenging with the physiological neurotransmitter acetylcholine (ACh); (2) separate blockade of mitochondrial Ca2+ uniporter (mCUP) with Ru360 or the mitochondrial Na+/Ca2+ exchanger (mNCX) with CGP37157; (3) full blockade of the mitochondrial Ca2+ cycling (mCC) by the simultaneous inhibition of the mCUP and the mNCX. Ru360 caused a pronounced delay of [Ca2+]c clearance and augmented secretion. In contrast, CGP37157 only caused a tiny delay of [Ca2+]c clearance and a mild decrease in secretion. The mCC resulting in continued Ca2+ uptake and its release back into the cytosol was interrupted by combined Ru360 + CGP37157 (Ru/CGP), the protonophore carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone, or combined oligomycin + rotenone (O/R); these three treatments caused a mild but sustained elevation of basal [Ca2+]c that, however, was not accompanied by a parallel increase in basal secretion. Nevertheless, all treatments caused a pronounced augmentation of ACh‐induced secretion, with minor changes of the ACh‐induced [Ca2+]c transients. Combined Ru/CGP did not alter the resting membrane potential in current‐clamped cells. Additionally, Ru/CGP did not increase basal [Ca2+]c near subplasmalemmal sites and caused a mild decrease in the size of the readily releasable vesicle pool. Our results provide new functional features in support of the view that in BCCs there are two subpopulations of mitochondria, M1 underneath the plasmalemma nearby exocytotic sites and M2 at the core cell nearby vesicle transport sites. While M1 serves to shape the ACh‐elicited exocytotic response through its efficient Ca2+ removal by the mCUP, M2 shapes the lower [Ca2+]c elevations required for new vesicle supply to the exocytotic machinery, from the large reserve vesicle pool at the cell core. The mCUP of the M1 pool seems to play a more prominent role in controlling the ACh responses, in comparison with the mNCX.

Mitochondrial Uncoupling: A Key Controller of Biological Processes in Physiology and Diseases

“…As a mitochondrial potential membrane is required for Ca 2+ entry into mitochondria [163], mitochondrial uncoupling will interfere with Ca 2+ homeostasis and with Ca 2+ -mediated protein exocytosis. In accordance with this theory, use of CCCP or FCCP decreases the Ca 2+ buffering capacity of mitochondria and increases global exocytosis in bovine [164,165] and rat chromaffin cells [166] but not in the same cell type in mice [167], suggesting species differences. In the rat insulinoma cell line INS-1E and in primary mouse and rat pancreatic beta cells, use of FCCP/CCCP inhibits glucose-stimulated insulin release (GSIS) [168,169,170].…”

“…For instance, FCCP leads to an increase in intracellular/cytosolic Ca 2+ concentration and to the secondary opening of Ca 2+ -activated K + channels, causing a plasma membrane hyperpolarization in mouse sensory neurons [77]. By decreasing the Ca 2+ buffering capacity of mitochondria, CCCP or FCCP increases global exocytosis in chromaffin cells [164,165,166]. In rat insulinoma cells and primary mouse and rat pancreatic beta cells, FCCP/CCCP inhibits glucose-stimulated (and Ca 2+ -dependent) insulin release (GSIS) [168,169,170].…”

The quantal catecholamine release from mouse chromaffin cells challenged with repeated ACh pulses is regulated by the mitochondrial Na+/Ca2+ exchanger

“… a ; Caricati‐Neto et al . ). Certainly, ACh and K + cause cell depolarisation to recruit calcium channels, thus stimulating Ca 2+ entry to evoke [Ca 2+ ] c transients, and the triggering of secretion (García et al .…”

“…However, in a later study we found that CGP diminished the [Ca 2+ ] c transients and catecholamine release triggered by ACh (Caricati‐Neto et al . ). This agrees with the decreased [Ca 2+ ] c transients (Fig.…”

Novel Purine Derivative ITH15004 Facilitates Exocytosis through a Mitochondrial Calcium-Mediated Mechanism

“…Decay of spike amplitudes may have various components affecting either Ca 2+ handling by the cell and/or vesicle transport and priming after each 35K + pulse, whereby the secretory machinery is depleted of a ready-release vesicle pool [ 3 ]. In BCCs, we have previously suggested that the lower [Ca 2+ ] c elevations required for vesicle transport and priming [ 24 ] is controlled by ER Ca 2+ fluxes [ 22 , 25 ] and/or by mitochondrial Ca 2+ movements [ 13 , 26 ]. As depletion of ER Ca 2+ with CRT did not affect the ability of ITH15004 to augment exocytosis ( Figure 3 B), it seemed that this Ca 2+ store is not contributing to the effects of the compound.…”