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

KEY POINTS: Upon repeated application of short ACh pulses to C57BL6J mouse chromaffin cells, the amperometrically monitored secretory responses promptly decayed to a steady-state level of around 25% of the initial response. A subsequent K+ pulse, however, overcame such decay. These data suggest that mouse chromaffin cells have a ready release-vesicle pool that is selectively recruited by the physiological neurotransmitter ACh. The ACh-sensitive vesicle pool is refilled and maintained by the rate of Ca2+ delivery from mitochondria to the cytosol, through the mitochondrial Na+ /Ca2+ exchanger (mNCX). ITH12662, a novel blocker of the mNCX, prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca2+ ]c clearance. This regulatory pathway may be physiologically relevant in situations of prolonged stressful conflicts where a sustained catecholamine release is regulated by mitochondrial Ca2+ circulation through the mNCX, which couples respiration and ATP synthesis to long-term stimulation of chromaffin cells by endogenously released ACh. ABSTRACT: Using caged-Ca2+ photorelease or paired depolarising pulses in voltage-clamped chromaffin cells (CCs), various pools of secretory vesicles with different readiness to undergo exocytosis have been identified. Whether these pools are present in unclamped CCs challenged with ACh, the physiological neurotransmitter at the splanchnic nerve-CC synapse, is unknown. We have explored here whether an ACh-sensitive ready-release vesicle pool (ASP) is present in C57BL6J mouse chromaffin cells (MCCs). Single cells were fast perfused with a Tyrode solution at 37°C, and challenged with 12 sequential ACh pulses (100 µm, 2 s, every 30 s) plus a K+ pulse given at the end (75 mm K+ ). After the first 2-3 ACh pulses the amperometrically monitored secretory responses promptly decayed to a steady-state level of around 25% of the initial response. The last K+ pulse, however, overcame such decay. Repeated ACh pulses to voltage-clamped cells elicited non-desensitising nicotinic currents. Also, the [Ca2+ ]c transients elicited by repeated ACh pulses that were superimposed on a stable baseline elevation did not undergo decay. The novel blocker of the mitochondrial Na+ /Ca2+ exchanger (mNCX) ITH12662 prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca2+ ]c clearance. The experiments are compatible with the idea that C57BL6J MCCs have an ASP vesicle pool that is selectively recruited by the physiological neurotransmitter ACh and is regulated by the rate of Ca2+ delivery from mitochondria to the cytosol, through the mNCX.

Faster kinetics of quantal catecholamine release in mouse chromaffin cells stimulated with acetylcholine, compared with other secretagogues.

Adrenal chromaffin cells (CCs) have been used extensively in studies aimed at revealing the intricacies of the Ca2+ -dependent early and late steps of regulated exocytosis. They have also served as invaluable models to study the kinetics of single-vesicle exocytotic events to infer the characteristics of opening and closing of the exocytotic fusion pore. We have here tested the hypothesis that stimulation at room temperature of CCs from mice C57BL/6 with physiological acetylcholine (ACh) and with other secretagogues (dimethylphenylpiperazinium, high K+ , muscarine, histamine, caffeine), alone or in combination, could trigger amperometric spike events with different kinetics. We found that mean secretory spike events in CCs stimulated with ACh had a fast rise rate of 25 pA/ms and a rapid decay time of 6.2 ms, with a small quantal size (0.31 pC). Surprisingly, these parameters considerably differed from those found in CCs stimulated with all other secretagogues that triggered secretory responses with spike events having smaller rise rates, longer decay times and higher quantal sizes. ACh spikes were unaltered by atropine but mitochondrial protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone markedly slowed down the rate rise and decay time, and augmented the quantal size of mean secretory events. We conclude that the physiological neurotransmitter ACh triggers a fast and efficient exocytotic response that cannot be mimicked by other secretagogues; such response is regulated by the mitochondrial circulation of calcium ions.